Methods for purification of single domain antigen binding molecules

ABSTRACT

Processes and methods of purifying or separating Single Domain Antigen Binding (SDAB) molecules that include one or more single binding domains (e.g., one or more nanobody molecules), substantially devoid of a complementary antibody domain and an immunoglobulin constant region, using Protein A-based affinity chromatography, are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 61/109,481, filed onOct. 29, 2008, the entire contents of which are hereby incorporated byreference in their entirety. This application also incorporates byreference the International Application filed with the U.S. ReceivingOffice on Oct. 29, 2009, entitled “Methods for Purification of SingleDomain Antigen Binding Molecules” and bearing attorney docket numberW2023-7038WO.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Oct. 28, 2009, is named982845_(—)1.txt, and is 10,967 bytes in size.

BACKGROUND

Recombinant proteins such as antibodies typically contain a variety ofimpurities that need to be removed before the protein product ispharmaceutically acceptable. Some of these impurities may include hostcell proteins (HCPs), DNA molecules, variant and/or misfolded forms ofthe product protein, and high molecular weight aggregates (HMWA). Theformation of aggregates is problematic during antibody production as itcan adversely affect product safety by causing complement activation oranaphylaxis upon administration. Aggregate formation may also hindermanufacturing processes by causing decreased product yield, peakbroadening, and loss of activity. These impurities can have a wide rangeof retention patterns on different modes of chromatography. Removal ofsuch broad spectrum of impurities is often difficult, typicallyrequiring multiple steps involving different modes of chromatography.

Common protein purification methods are predicated on differences in thesize, charge, and solubility between the protein to be purified and thecontaminants. Protocols based on these parameters include, but are notlimited to, affinity chromatography, ion exchange chromatography, sizeexclusion chromatography, and hydrophobic interaction chromatography.These chromatographic methods, however, sometimes present technicaldifficulties in the separation of aggregated or multimeric species ofantibodies. Techniques such as ion exchange and hydrophobic interactionchromatography, for example, may induce the formation of aggregates dueto an increased protein concentration or the required changes in bufferconcentration and/or pH during elusion. Further, in several instancesantibodies show differences in isoelectric points that are too small toallow for their separation by ion-exchange chromatography (Tarditi, J.Immunol. Methods 599:13-20 (1992)). Size exclusion chromatography tendsto be cumbersome and results in the significant dilution of the product,which is a hindrance in large-scale, efficiency-based manufacturingprocesses. Leakage of ligands from affinity chromatography columns canalso occur, which results in undesirable contamination of the elutedproduct (Steindl, J. Immunol. Methods 235:61-69 (2000)).

While several different modalities of chromatography can be employedduring the purification of recombinant proteins, the need still existsto develop purification processes that reduce the number ofchromatography steps used and that do not destroy, or significantlyreduce, the biological activity of the recombinant protein.

SUMMARY

The present invention is based, in part, on the discovery that singledomain antigen binding (SDAB) molecules interact with, e.g., bind to,Protein A or a functional variant thereof, thereby enabling the use ofProtein A-based affinity chromatography in the purification of the SDABmolecules. In other embodiments, the SDAB molecules can be purifiedusing other chromatographic techniques, such as ion (e.g., cation)exchange chromatography. The SDAB molecule can include one or moresingle antigen binding domains that interact with, e.g., bind to, one ormore target proteins (e.g., tumor necrosis factor and/or human serumalbumin). In certain embodiments, the SDAB molecule is a single chainpolypeptide comprised of one or more nanobody molecules, beingsubstantially devoid of a complementary antibody domain and/or animmunoglobulin constant region. Thus, the present invention relates toprocesses and methods of purifying or separating SDAB molecules thatinclude one or more single binding domains (e.g., one or more nanobodymolecules), using chromatographic techniques such as Protein A-basedaffinity chromatography and ion (e.g., cation) exchange chromatography,individually or in combination.

[Note: Nanobody™ and Nanobodies™ are Registered Trademarks of AblynxN.V.]

Accordingly, in one aspect, the invention features a method, or process,of separating or purifying an SDAB molecule (e.g., one or more nanobodymolecules) from a mixture containing the SDAB molecule and one or morecontaminants (also referred to herein as an “SDAB moleculepreparation”). The method or process includes: contacting the mixturewith a Protein A-based support or an ion (e.g., cation) exchange (CEX)support, under conditions that allow the SDAB molecule to bind or absorbto the support; removing one or more contaminants, e.g., by washing thebound support under conditions where the SDAB molecule remains bound tothe support (e.g., washing the bound support with at least one Protein Aor CEX washing buffer); and selectively eluting the SDAB molecule fromthe support, e.g., by eluting the adsorbed SDAB molecule with at leastone Protein A or CEX elution buffer.

In one embodiment, the method of separating or purifying the SDABmolecule includes contacting the mixture of the SDAB molecule and one ormore contaminants with a cation exchange support.

In other embodiments, the method of separating or purifying the SDABmolecule includes contacting the mixture of the SDAB molecule and one ormore contaminants with a Protein A-based resin.

The method or process can be used alone, or in combination with, atleast one other purification method, including, but not limited to, oneor more of: hydroxyapatite, affinity chromatography, size exclusionchromatography, hydrophobic interaction chromatography, metal affinitychromatography, diafiltration, ultrafiltration, viral inactivation(e.g., using low pH) and/or viral removal filtration. For example, themethod or process can be used in combination with one or more ofhydroxyapatite chromatography, ultrafiltration, viral inactivation(e.g., using low pH) and/or viral removal filtration. In embodimentswhere a Protein A-support is used, the method or process can furtherinclude ion (e.g., cation or anion) exchange chromatography.

In embodiments, the method or process further includes contacting themixture with a hydroxyapatite resin and selectively eluting the SDABmolecule from the hydroxyapatite resin. In other embodiments where aProtein A-support is used, the method or process further includescontacting the mixture with a cation exchange (CEX) column, andselectively eluting the SDAB molecule from the column.

Embodiments of the aforesaid methods and processes may include one ormore of the following features:

In one embodiment, the SDAB molecule separated or purified by the methodor process of the invention is a recombinant protein produced as aproduct of a cell culture, e.g., a host cell (e.g., a mammalian, e.g., aChinese Hamster Ovary (CHO), cell) in a mixture that includes the SDABmolecule and cell culture contaminants. The cell culture can be a smallor a large scale culture.

In other embodiments, the contaminants in the mixture separated orpurified by the method or process of the invention include one or moreof high molecular weight protein aggregates, host cell proteins, DNA,and/or Protein A (e.g., leached protein A). In embodiments, the SDABmolecule is purified to at least 85%, 90%, 95%, 96%, 97%, 98%, 99% orhigher purity.

In another embodiment, the Protein A-based support used in the method orprocess of the invention includes a support, e.g., a resin, ofimmobilized Protein A (e.g., recombinant or isolated Protein A), or afunctional variant thereof. In one embodiment, the immobilized Protein Ais full length Staphylococcal Protein A (SpA) composed of five domainsof about 50-60 amino acid residues known as E, D, A, B and C domains inorder from the N-terminus. For example, the Protein A includes the aminoacid sequence of SpA (SEQ ID NO:11) shown in FIG. 4A, or an amino acidsequence substantially identical thereto (e.g., an amino acid sequenceat least 85%, 90%, 95% or more identical to the amino acid sequence ofSEQ ID NO:11 shown in FIG. 4A). In other embodiments, the immobilizedProtein A is a functional variant of SpA that includes at least onedomain chosen from E, D, A, B and/or C, or a modified form thereof. Forexample, the functional variant of SpA can include at least domain B ofSpA, or a variant of domain B, having one or more substituted asparagineresidues, also referred to herein as domain Z. In one embodiment, thefunctional variant of SpA includes the amino acid sequence of SEQ IDNO:12 shown in FIG. 4B, or an amino acid sequence substantiallyidentical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%or more identical to the amino acid sequence of SEQ ID NO:12 shown inFIG. 4B). Other permutations of functional variants of Protein A can beused comprising domain B, or a variant domain B, and one or more of:domains A and/or C; domains E, A and/or C; or domains E, D, A and/or C.Any combination of E, D, A, B and/or C, or a functional variant thereof,can be used as long as the combination is capable of binding to the SDABmolecule. Exemplary Protein A support resins that can be used includeMabSELECT™ columns, MabSELECT™ SuRe columns, MabSELECT™ Xtra (GEHealthcare Products), and ProSep™ Va Ultra Plus (Millipore Corporation,Billerica Mass.).

In one embodiment where a Protein A-based support is used in the methodor process of the invention, the mixture of SDAB molecules andcontaminants are contacted with, e.g., loaded onto, the Protein A-basedsupport under conditions that allow the SDAB molecule to bind or absorbto the Protein A-based support. In certain embodiments, a Protein Aloading buffer is used that includes a conditioned medium. The Protein-Acolumn can be equilibrated using a Protein A equilibration solution thatincludes about 10 to about 250 mM NaCl and about 10 to about 100 mM Trisat pH ranging from about 6 to 8; about 50 to about 200 mM NaCl and about20 to about 75 mM Tris at pH ranging from about 6.5 to 7.5; about 100 toabout 175 mM NaCl and about 40 to about 60 mM Tris at pH ranging fromabout 7 to 7.5; about 125 to about 160 mM NaCl and about 45 to about 55mM Tris at pH ranging from about 7 to 7.5; about 50 to about 150 mM NaCland about 50 mM Tris at pH ranging from about 7.5; or about 150 mM NaCland about 50 mM Tris at pH ranging from about 6.5, 7.0, 7.5, or 8.0.

In yet another embodiment where a Protein A-based support is used in themethod or process of the invention, one or more contaminants of themixture are removed, e.g., by washing the bound support under conditionswhere the SDAB molecule remains bound to the support (e.g., washing thebound support with at least one Protein A washing buffer). In certainembodiments, the Protein A washing buffer includes includes about 10 toabout 250 mM NaCl and about 10 to about 100 mM Tris at pH ranging fromabout 6 to 8; about 50 to about 200 mM NaCl and about 20 to about 75 mMTris at pH ranging from about 6.5 to 7.5; about 100 to about 175 mM NaCland about 40 to about 60 mM Tris at pH ranging from about 7 to 7.5;about 125 to about 160 mM NaCl and about 45 to about 55 mM Tris at pHranging from about 7 to 7.5; about 50 to about 150 mM NaCl and about 50mM Tris at pH ranging from about 7.5; or about 150 mM NaCl and about 50mM Tris at pH ranging from about 6.5, 7.0, 7.5, or 8.0. In someembodiments, the washing buffer includes 50 mM NaCl and 50 mM Tris at pH7.5. In some embodiments, the washing buffer includes 10 mM, 25 mM, 50mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450mM, or 500 mM NaCl. In some embodiments, the washing buffer includes 10mM, 25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM,400 mM, 450 mM, or 500 mM CaCl₂. In some embodiments, the washing bufferincludes 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300mM, 350 mM, 400 mM, 450 mM, or 500 mM Tris. In some embodiments, thewashing buffer includes 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 150 mM, 200mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM Citrate. In someembodiments, the washing buffer includes 10 mM, 25 mM, 50 mM, 75 mM, 100mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mMHEPES. In some embodiments, the washing buffer is at pH 6.0, 6.5, 7.0,7.5, 8.0, 8.5 or 9.0.

In yet another embodiment where a Protein A-based support is used in themethod or process of the invention, the SDAB molecule is selectivelyeluted from the support, e.g., by eluting the adsorbed SDAB moleculewith at least one Protein A elution buffer. In some embodiments, theelution buffer includes about 5 to about 50 mM NaCl and about 5 mM toabout 100 mM glycine at pH 4.0 or less. In some embodiments, the elutionbuffer includes about 10 mM, about 25 mM, about 50 mM, about 75 mM,about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM,about 350 mM, about 400 mM, about 450 mM, or about 500 mM NaCl; about 10mM, about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 150 mM,about 200 mM, or about 250 mM glycine. In some embodiments, the elutionbuffer is at pH 2.0, 2.5, 3.0, 3.5, or 4.0. In certain embodiments, theProtein A eluting buffer includes about 10 mM NaCl and about 50 mMglycine at about pH 3.0.

In one embodiment, ceramic hydroxyapatite chromatography is used incombination with Protein A chromatography in the method or process ofthe invention. The ceramic hydroxyapatite chromatography can be usedprior to, or more frequently after, the Protein-A based chromatography.In such embodiments, the method includes contacting the mixture of theSDAB molecule (e.g., the mixture after separation or purification withProtein A chromatography) with a hydroxyapatite resin and selectivelyeluting the SDAB molecule from the resin. Alternatively the mixture maybe pre-treated with an equilibration buffer and then allowed to flowthrough a hydroxyapatite resin. Either of these methods may also be usedin combination to purify the mixtures. In one embodiment, the elutionand load buffers include about 1 to about 20 mM sodium phosphate andfrom about 0.2 to about 2.5 M sodium chloride, wherein the elution andload buffers have a pH from about 6.4 to about 7.6. In otherembodiments, the equilibration buffer and wash buffer include about 1 toabout 20 mM sodium phosphate, from about 0.01 to about 2.0 M sodiumchloride, from about 0 to about 200 mM arginine, and from about 0 toabout 200 mM HEPES, wherein the equilibration and wash buffers have a pHfrom about 6.2 to 8.0. In embodiments, the resulting purified SDABmolecule contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% orless high molecular weight aggregates.

In other embodiments, the ion exchange chromatography is used incombination with one or both of Protein A chromatography and/orhydroxyapatite chromatography as described herein. An exemplary methodor process where the Protein A chromatography is carried out before theion exchange chromatography includes: contacting the mixture containingthe SDAB molecule and one or more contaminants with a Protein A support,allowing the SDAB molecule to adsorb to the support, washing the supportand adsorbed SDAB molecule with at least one Protein A washing buffer,eluting the adsorbed SDAB molecule with at least one Protein A elusionbuffer, thereby collecting an SDAB molecule preparation. The method orprocess can further include contacting the SDAB molecule preparationwith an ion exchange support, allowing the SDAB molecule to flow throughthe support, washing the support with at least one ion exchange washingbuffer, thereby collecting the ion exchange flow-through. In certainembodiments, the method or process further includes contacting the ionexchange flow-through with a hydroxyapatite resin, allowing theflow-through to adsorb to the resin, washing the resin with at least onehydroxyapatite washing buffer, and eluting purified SDAB molecule fromthe resin with at least one hydroxyapatite elusion buffer.

In other embodiments, ion (e.g., cation) exchange chromatography (CEX)is used alone, or in combination with another resin, e.g., one or bothof Protein A chromatography and/or ceramic hydroxyapatitechromatography. The method or process includes contacting the mixturecontaining the SDAB molecule and one or more contaminants with an ionexchange support, allowing the SDAB molecule to flow through thesupport, washing the support with at least one ion (e.g., cation)exchange washing buffer. In one embodiment, cation exchange support ischosen from: Capto™ S (GE Heathcare), Fractogel® SO3-(M) (EMDChemicals), Toyopearl® Gigacap S-650M (Tosoh Bioscience) or Poros® HS 50(Applied Biosystems). In one embodiment, the CEX resin shows a capacityof at least about 10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, or 60 g/L. Inother embodiments, the conductivity of the condition media (CM) used toload the column is between about 15 and 5 mS/cm, 14 and 6 mS/cm, 13 and8 mS/cm, 12 and 9 mS/cm, or 11 to 10 mS/cm, or about 7 mS/cm, 8 mS/cm, 9mS/cm, 10 mS/cm, 11 mS/cm, 12 mS/cm, or 13 mS/cm. In other embodiments,the pH of the loading conditions is adjusted to less than about 6, 5.5,5, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9, 3.8, or 3.7. In embodiments, theelution buffer is about 100 mM sodium chloride or less, about 90 mMsodium chloride or less, about 80 mM sodium chloride or less, about 70mM sodium chloride or less, about 60 mM sodium chloride or less, about50 mM sodium chloride or less, about 40 mM sodium chloride or less, orabout 30 mM sodium chloride or less, 20 mM sodium chloride or less,about 10 mM sodium chloride or less, about 5 mM sodium chloride or less,about 1 mM sodium chloride or less, and has a pH of about 4 to 8, about5 to 7.5, about 5.5 to 7.2, about 6 to 7.1, or about 6.5 to 7, or about5, 5, 5, 6, 6, 5, or 7. In other embodiments, the CEX column could alsobe eluted using the downstream cHA equilibration buffer.

In certain embodiments, the cation exchange chromatography is the onlychromatographic method used in the SDAB purification. In otherembodiments, cation exchange chromatography is used in combination withother chromatographic methods (e.g., hydroxyapatite chromatography). Anexemplary method or process where the cation exchange chromatography iscarried out includes: contacting the mixture containing the SDABmolecule and one or more contaminants with a cation exchange supportunder conditions that reduce the conductivity of the loading buffer orconditioned medium (e.g., under conditions about 15 and 5 mS/cm, 14 and6 mS/cm, 13 and 8 mS/cm, 12 and 9 mS/cm, or 11 to 10 mS/cm, or about 7mS/cm, 8 mS/cm, 9 mS/cm, 10 mS/cm, 11 mS/cm, 12 mS/cm, or 13 mS/cm),allowing the SDAB molecule to adsorb to the support, washing the supportand adsorbed SDAB molecule with at least one cation exchange washingbuffer, eluting the adsorbed SDAB molecule with at least one elusionbuffer, thereby collecting an SDAB molecule preparation. The method orprocess can further include contacting the SDAB molecule preparationwith another support or resin, for example, the method or process canfurther include contacting the ion exchange flow-through with ahydroxyapatite resin, allowing the flow-through to adsorb to the resin,washing the resin with at least one hydroxyapatite washing buffer, andeluting purified SDAB molecule from the resin with at least onehydroxyapatite elusion buffer.

In other embodiments, the method or process further includesconcentrating the eluted SDAB molecule, e.g., by performing anultrafiltration/diafiltration step, to a preset target volume. Theconcentration step can also be used to exchange the buffer of the elutedSDAB molecule. For example, the concentrated, eluted SDAB molecule canbe filtered, e.g., diafiltered, in the presence of a Histidine buffer ora Tris buffer. In embodiments where the Histidine buffer is used, thebuffer is at a concentration of at least about 5 to 30 mM, about 7.5 to28 mM, about 10 to 20 mM, about 12 to 15 mM, or about 10 mM, about 11mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 20 mM,about 25 mM, about 28 mM at a pH of about 7, about 6, about 5, about 4,about 3, or in the range of about 4 to 6.5, about 5 to 6, about 5.9,about 5.8, about 5.7, about 5.6, or about 5.5. In embodiments, a smallvolume of concentrated formulation buffer is added to the eluted,concentrated SDAB molecule (e.g., at least 2, 5, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 20% v/v of the concentrated formulation buffer. Inembodiments, the concentrated formulation buffer is about 10 to 50 mMHistidine (e.g., about 20 mM, about 30 mM Histidine, about 10 to 60%sugar (e.g., sucrose, sorbitol or trehalose), e.g., about 50% sucrose,and a surfactant (e.g., polysorbate 80) at about 0.001 to about 0.1%,e.g., about 0.06%). Exemplary formulations for the SDAB molecules aredescribed in U.S. Ser. No. 12/608,553, filed on Oct. 29, 2008 in thename of Wyeth, the contents of which are incorporated by referenceherein.

In embodiments, the SDAB molecule is concentrated to at least about 20g/L, 30 g/L, 40 g/L, 80 g/L, 90 g/L g/L, 100 g/L, 150 g/L, 200 g/L, 210g/L, 220 g/L, 230 g/L, 240 g/L, 250 g/L, 260 g/L, 270 g/L, 280 g/L, 290g/L, 300 g/L, 310 g/L, 320 g/L, 330 g/L, 340 g/L, 350 g/L or higher.

In certain embodiments, the method or process includes: evaluating(e.g., detecting, quantifying and/or monitoring) at least one parameterof the purity, activity, toxicity, pharmacokinetics and/orpharmacodynamics of the SDAB molecule; (optionally) comparing the atleast one parameter with a reference value, to thereby evaluate orselect the SDAB molecule. The comparison can include determining if theat least one parameter has a pre-selected relationship with thereference value, e.g., determining if it falls within a range of thereference value (either inclusive or exclusive of the endpoints of therange); is equal to or greater than the reference value. In certainembodiments, if the at least one parameter meets a pre-selectedrelationship, e.g., falls within the reference value, the SDAB moleculeis selected. In other embodiments, the assays, methods, or an indicationof whether the pre-selected relationship between the at least oneparameter and a reference value is met, is recorded or memorialized,e.g., in a computer readable medium. Such methods, assays or indicationsof meeting pre-selected relationship can be listed on the productinsert, a compendium (e.g., the U.S. Pharmacopeia), or any othermaterials, e.g., labeling that may be distributed, e.g., for commercialuse, or for submission to a U.S. or foreign regulatory agency.

In one embodiment, the method or process further includes comparing thevalue determined with a reference value, to thereby analyze themanufacturing process.

In one embodiment, the method further includes maintaining themanufacturing process based, at least in part, upon the analysis. In oneembodiment, the method further includes altering the manufacturingprocess based upon the analysis.

In another embodiment the method includes evaluating a process, e.g.,manufacturing process, of the SDAB molecule, e.g., a TNF nanobodymolecule, made by a selected process, that includes making adetermination about the process based upon a method or analysisdescribed herein. In one embodiment, the method further includesmaintaining or altering the manufacturing process based, at least inpart, upon the method or analysis. Thus, in another embodiment the partymaking the evaluation does not practice the method or analysis describedherein but merely relies on results which are obtained by a method oranalysis described herein.

In another embodiment the method includes comparing two or morepreparations in a method of monitoring or controlling batch-to-batchvariation or to compare a preparation to a reference standard.

In yet another embodiment, the method can further include making adecision, e.g., to classify, select, accept or discard, release orwithhold, process into a drug product, ship, move to a differentlocation, formulate, label, package, release into commerce, sell oroffer for sale the preparation, based, at least in part, upon thedetermination.

In another aspect, the invention features a method of complying with aregulatory requirement, e.g., a post approval requirement of aregulatory agency, e.g., the FDA. The method includes providing anevaluation of a parameter of SDAB molecule, as described herein. Thepost approval requirement can include a measure of one more of the aboveparameters. The method also includes, optionally, determining whetherthe observed solution parameter meets a preselected criteria or if theparameter is in a preselected range; optionally, memorializing the valueor result of the analysis, or communicating with the agency, e.g., bytransmitting the value or result to the regulatory agency.

In another aspect, the invention features a method of one or more of:providing a report to a report-receiving entity, evaluating a sample ofan SDAB molecule, e.g., a TNF nanobody molecule, for compliance with areference standard, e.g., an FDA requirement, seeking indication fromanother party that a preparation of the SDAB molecule meets somepredefined requirement, or submitting information about a preparation ofan SDAB molecule to another party. Exemplary receiving entities or otherparties include a government, e.g., the U.S. federal government, e.g., agovernment agency, e.g., the FDA. The method includes one or more (orall) of the following steps for making and/or testing the SDAB moleculein a first country, e.g., the U.S.; sending at least an aliquot of thesample outside the first country, e.g., sending it outside the UnitedStates, to a second country; preparing, or receiving, a report whichincludes data about the structure of the preparation of the SDABmolecule, e.g., data related to a structure and/or chain describedherein, e.g., data generated by one or more of the methods describedherein; and providing said report to a report recipient entity.

The SDAB molecule, e.g., the nanobody molecule (e.g., the TNF-bindingnanobody molecule) separated or purified by the method or process of theinvention can include one or more single binding domains (e.g., one ormore nanobodies). For example, the nanobody molecule can comprise, orconsist of, a polypeptide, e.g., a single chain polypeptide, comprisingat least one immunoglobulin variable domain (including one, two or threecomplementarity determining regions (CDRs)). Examples of SDAB moleculesinclude molecules naturally devoid of light chains (e.g., VHH,nanobodies, or camelid derived antibodies). Such SDAB molecules can bederived or obtained from camelids such as camel, llama, dromedary,alpaca and guanaco. In other embodiments, the SDAB molecule may includesingle domain molecules including, but not limited to, othernaturally-occurring single domain molecules, such as shark single domainpolypeptides (IgNAR); and single domain scaffolds (e.g., fibronectinscaffolds). Single domain molecules may be derived from shark.

In one embodiment, the SDAB molecule separated or purified by the methodor process of the invention is a single chain polypeptide comprised ofone or more single domain molecules. In embodiments, the nanobodymolecule is monovalent or multivalent (e.g., bivalent, trivalent, ortetravalent). In other embodiments, the nanobody molecule ismonospecific or multispecific (e.g., bispecific, trispecific ortetraspecific). The SDAB molecule may comprise one or more single domainmolecules that are recombinant, CDR-grafted, humanized, camelized,de-immunized, and/or in vitro generated (e.g., selected by phagedisplay). For example, the SDAB molecule can be a single chain fusionpolypeptide comprising one or more single domain molecules that binds toone or more target antigens. Typically, the target antigen is amammalian, e.g., a human protein. In certain embodiments, the SDABmolecule binds to a serum protein, e.g., a human serum proteins chosenfrom one or more of serum albumin (human serum albumin (HSA)), fibrin,fibrinogen, or transferrin.

In one exemplary embodiment, the SDAB molecule separated or purified bythe method or process of the invention is a trivalent, bispecificmolecule composed of a single chain polypeptide fusion of two singledomain molecules (e.g., two camelid variable regions) that bind to atarget antigen, e.g., tumor necrosis factor α (TNF α), and one singledomain molecule (e.g., a camelid variable region) that binds to a serumprotein, e.g., HSA. The single domain molecules of the SDAB molecule canbe arranged in the following order from N- to C-terminus: TNFα-bindingsingle domain molecule—HAS-binding single domain molecule—TNFα-bindingsingle domain molecule. It will be appreciated that any order orcombination of single domain molecules against one or more targets canbe formulated as described herein.

In one embodiment, the SDAB molecule separated or purified by the methodor process of the invention is referred to herein as “ATN-103,”comprises, or consists of, the amino acid sequence of SEQ ID NO:1 shownin FIG. 2, or an amino acid sequence substantially identical thereto(e.g., an amino acid sequence at least 85%, 90%, 95% or more identicalto the amino acid sequence of SEQ ID NO:1 shown in FIG. 2). Examples ofadditional trivalent, bispecific nanobody molecules that can beformulated as described herein include TNF24, TNF25, TNF26, TNF27,TNF28, TNF60 and TNF62 disclosed in Table 29 of WO 2006/122786.

In certain embodiments, at least one of the single domain molecule ofthe SDAB molecule separated or purified by the method or process of theinvention binds to TNFα includes one, two, or three CDRs having theamino sequence: DYWMY (CDR1), EINTNGLITKYPDSVKG (CDR2) and/or SPSGFN(CDR3), or having a CDR that differs by fewer than 3, 2 or 1 amino acidsubstitutions (e.g., conservative substitutions) from one of said CDRs.In other embodiments, the single domain molecule comprises a variableregion having the amino acid sequence from about amino acids 1 to 115 ofSEQ ID NO:1 shown in FIG. 2, or an amino acid sequence substantiallyidentical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%or more identical to the amino acid sequence of SEQ ID NO:1 shown inFIG. 2). In embodiments, the TNFα-binding single domain molecule has oneor more biological activities of the TNFα-binding single domain antibodymolecule of SEQ ID NO:1 shown in FIG. 2. For example, the TNFα-bindingsingle domain molecule binds to the same or a similar epitope as theepitope recognized by the TNFα-binding single domain molecule of SEQ IDNO:1 shown in FIG. 2 (e.g., binds to TNFα in its trimeric form; binds tothe TNFα site contacting the TNF receptor; binds to an epitope in theTNFα trimer comprising Gln at position 88 and Lys at position 90 on thefirst TNF monomer (monomer A), and Glu at position 146 on the second TNFmonomer (monomer B), or an epitope as disclosed in WO 06/122786). Inother embodiment, the TNFα-binding single domain molecule has anactivity (e.g., binding affinity, dissociation constant, bindingspecificity, TNF-inhibitory activity) similar to any of the TNFα-bindingsingle domain molecule disclosed in WO 06/122786.

In other embodiments, the TNFα-binding nanobody molecule comprises oneor more of the nanobodies disclosed in WO 2006/122786. For example, theTNFα-binding nanobody molecule can be a monovalent, bivalent, trivalentTNFα-binding nanobody molecule disclosed in WO 2006/122786. ExemplaryTNFα-binding nanobodies include, but are not limited to, TNF1, TNF2,TNF3, humanized forms thereof (e.g., TNF29, TNF30, TNF31, TNF32, TNF33).Additional examples of monovalent TNFα-binding nanobodies are disclosedin Table 8 of WO 2006/122786. Exemplary bivalent TNFα-binding nanobodymolecules include, but are not limited to, TNF55 and TNF56, whichcomprise two TNF30 nanobodies linked via a peptide linker to form asingle fusion polypeptide (disclosed in WO 2006/122786). Additionalexamples of bivalent TNFα-binding nanobody molecules are disclosed inTable 19 of WO 2006/122786 as TNF4, TNF5, TNF6, TNF7, TNF8).

In other embodiments, at least one of the single domain molecule of theSDAB molecule separated or purified by the method or process of theinvention binds to HSA includes one, two, or three CDRs having the aminosequence: SFGMS (CDR1), SISGSGSDTLYADSVKG (CDR2) and/or GGSLSR (CDR3),or having a CDR that differs by fewer than 3, 2 or 1 amino acidsubstitutions (e.g., conservative substitutions) from one of said CDRs.In other embodiments, the single domain molecule comprises a variableregion having the amino acid sequence from about amino acids 125 to 239of SEQ ID NO:1 shown in FIG. 2, or an amino acid sequence substantiallyidentical thereto (e.g., an amino acid sequence at least 85%, 90%, 95%or more identical to the amino acid sequence of SEQ ID NO:1 shown inFIG. 2). In embodiments, the HSA-binding single domain molecule has oneor more biological activities of the HSA-binding single domain moleculeof SEQ ID NO:1 shown in FIG. 2. For example, the HSA-binding singledomain molecule binds to the same or a similar epitope as the epitoperecognized by the HSA-binding single domain molecule of SEQ ID NO:1shown in FIG. 2. In other embodiment, the HSA-binding single domainmolecule has an activity (e.g., binding affinity, dissociation constant,binding specificity) similar to any of the HSA-binding single domainmolecule disclosed in WO 06/122786.

In other embodiments, the HSA-binding SDAB molecule comprises one ormore of the nanobodies disclosed in WO 2006/122786. For example, theHSA-binding SDAB molecule can be a monovalent, bivalent, trivalentHSA-binding nanobody molecule disclosed in WO 2006/122786. In otherembodiments, the HSA-binding SDAB molecule can be a monospecific or amultispecific molecule having at least one of the binding specificitiesbind to HSA. Exemplary TNFα-binding nanobodies include, but are notlimited to, ALB1, humanized forms thereof (e.g., ALB6, ALB7, ALB8, ALB9,ALB10), disclosed in WO 06/122786.

In other embodiments, two or more of the single domain molecules of theSDAB molecules are fused, with or without a linking group, as a geneticor a polypeptide fusion. The linking group can be any linking groupapparent to those of skill in the art. For instance, the linking groupcan be a biocompatible polymer with a length of 1 to 100 atoms. In oneembodiment, the linking group includes or consists of polyglycine,polyserine, polylysine, polyglutamate, polyisoleucine, or polyarginineresidues, or a combination thereof. For example, the polyglycine orpolyserine linkers can include at least five, seven eight, nine, ten,twelve, fifteen, twenty, thirty, thirty-five and forty glycine andserine residues. Exemplary linkers that can be used include Gly-Serrepeats, for example, (Gly)₄-Ser repeats of at one, two, three, four,five, six, seven or more repeats (SEQ ID NO:8). In embodiments, thelinker has the following sequences: (Gly)₄-Ser-(Gly)₃-Ser or((Gly)₄-Ser)n, where n is 4, 5, or 6 (SEQ ID NO:10).

The SDAB molecule separated or purified by the method or process of theinvention can be further modified by associating, e.g., covalently ornon-covalently a second moiety. For example, the nanobody molecule canbe covalently attached to a suitable pharmacologically acceptablepolymer, such as poly(ethyleneglycol) (PEG) or a derivative thereof(such as methoxypoly(ethyleneglycol) or mPEG). Examples of pegylatednanobody molecules are disclosed as TNF55-PEG40, TNF55-PEG60,TNF56-PEG40 and TNF56-PEG60 in WO 06/122786.

In one embodiment, the method or process further comprises one or moreof ion (e.g., cation or anion) exchange chromatography, hydroxyapatitechromatography, affinity chromatography, size exclusion chromatography,hydrophobic interaction chromatography, metal affinity chromatography,diafiltration, ultrafiltration, and/or viral removal filtration.

In one embodiment, the method or process further includes preparing aformulation of the recombinant SDAB molecule as a pharmaceuticalcomposition. The formulation can include the SDAB molecule alone or incombination with a second agent, e.g., a second therapeutically orpharmacologically active agent that is useful in treating a TNFαassociated disorder, e.g., inflammatory or autoimmune disorders,including, but not limited to, rheumatoid arthritis (RA) (e.g., moderateto severe rheumatoid arthritis), arthritic conditions (e.g., psoriaticarthritis, polyarticular juvenile idiopathic arthritis (JIA), ankylosingspondylitis (AS), psoriasis, ulcerative colitis, Crohn's disease,inflammatory bowel disease, and/or multiple sclerosis. For example, thesecond agent may be an anti-TNF antibody or TNF binding fragmentthereof, wherein the second TNF antibody binds to a different epitopethan the TNF-binding SDAB molecule of the formulation. Othernon-limiting examples of agents that can be co-formulated with theTNF-binding SDAB molecule include, but are not limited to, a cytokineinhibitor, a growth factor inhibitor, an immunosuppressant, ananti-inflammatory agent, a metabolic inhibitor, an enzyme inhibitor, acytotoxic agent, and a cytostatic agent. In one embodiment, theadditional agent is a standard treatment for arthritis, including, butnot limited to, non-steroidal anti-inflammatory agents (NSAIDs);corticosteroids, including prednisolone, prednisone, cortisone, andtriamcinolone; and disease modifying anti-rheumatic drugs (DMARDs), suchas methotrexate, hydroxychloroquine (Plaquenil) and sulfasalazine,leflunomide (Arava®), tumor necrosis factor inhibitors, includingetanercept (Enbrel®), infliximab (Remicade®) (with or withoutmethotrexate), and adalimumab (Humira®), anti-CD20 antibody (e.g.,Rituxan®), soluble interleukin-1 receptor, such as anakinra (Kineret®),gold, minocycline (Minocin®), penicillamine, and cytotoxic agents,including azathioprine, cyclophosphamide, and cyclosporine. Suchcombination therapies may advantageously utilize lower dosages of theadministered therapeutic agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies.

In another aspect, the invention features an SDAB molecule made by themethod or process described herein. Compositions, e.g., pharmaceuticalcompositions and formulations, containing the SDAB molecules made by themethod or process described herein are also encompassed by thisinvention. For example, the formulations may include the SDAB moleculesdescribed herein in a pharmaceutically acceptable carrier.

In one embodiment, the SDAB molecules made by method or processdescribed herein are suitable for administration to a subject, e.g., ahuman subject (e.g., a patient having a TNFa associated disorder). Forexample, the SDAB molecule or formulation thereof can be administered tothe subject by injection (e.g., subcutaneous, intravascular,intramuscular or intraperitoneal) or by inhalation.

In another aspect, the invention relates to methods for treating orpreventing in a subject (e.g., a human subject) a disorder associatedwith an SDAB molecule described herein (e.g., a TNFa-associateddisorder, e.g., inflammatory or autoimmune disorders, including, but notlimited to, rheumatoid arthritis (RA) (e.g., moderate to severerheumatoid arthritis), arthritic conditions (e.g., psoriatic arthritis,polyarticular juvenile idiopathic arthritis (JIA), ankylosingspondylitis (AS), psoriasis, ulcerative colitis, Crohn's disease,inflammatory bowel disease, and/or multiple sclerosis). The methodincludes administering to a subject, e.g., a human patient, apharmaceutical composition includes a TNF-binding SDAB made by themethod or process described herein, alone or in combination with any ofthe combination therapies described herein, in an amount such that oneor more of the symptoms of the TNFα associated disorder are reduced.

In another aspect, the invention features a kit or an article ofmanufacture that includes a device, a syringe or a vial containing theSDAB made by the method or process described herein.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic diagram of the predicted structure ofATN-103.

FIG. 2 depicts the amino acid sequence of ATN-103 polypeptide chain (SEQID NO:1).

FIG. 3 depicts a flow diagram of the ATN-103 purification process.

FIG. 4A depicts the amino acid sequence of full length StaphylococcalProtein A (SpA) (SEQ ID NO:11). FIG. 4B depicts the amino acid sequenceof modified domain B of SpA (SEQ ID NO:12). The α-helix regions areindicated in bold.

DETAILED DESCRIPTION

The present invention is based, at least in part, on the discovery thatan SDAB molecule that includes one or more single binding domains (e.g.,one or more nanobody molecules) interacts with, e.g., binds to, ProteinA or a functional variant thereof, thereby enabling the use of ProteinA-based affinity modalities of chromatography in the purification of theSDAB molecule. Thus, the present invention relates to processes andmethods of purifying or separating antigen-binding fusion polypeptidesthat include one or more single binding domains (e.g., one or morenanobody molecules), devoid of a complementary antibody domain and animmunoglobulin Fc region, using Protein A-based affinity chromatography.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

The terms “proteins” and “polypeptides” are used interchangeably herein.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

The term “SDAB molecule preparation” refers to any compositioncontaining a SDAB molecule and/or one or more unwanted contaminants. Thepreparation may be partially separated or purified, e.g., by passingthrough a chromatographic column as described herein, e.g., a ProteinA-based or cation exchange support.

The term “chromatography” refers to the separation of chemicallydifferent molecules in a mixture from one another by contacting themixture with an adsorbent, wherein one class of molecules reversiblybinds to or is adsorbed onto the adsorbent. Molecules that are leaststrongly adsorbed to or retained by the adsorbent are released from theadsorbent under conditions where those more strongly adsorbed orretained are not.

The term “flow-through mode” refers to an SDAB molecule preparationseparation technique in which at least one SDAB molecule contained inthe preparation is intended to flow through a chromatographic resin orsupport, while at least one potential contaminant or impurity binds tothe chromatographic resin or support. Flow-through mode may be used, forinstance, in hydroxyapatite chromatography and ion exchangechromatography.

“Binding mode” refers to an SDAB molecule preparation separationtechnique in which at least one antibody molecule contained in thepreparation binds to a chromatographic resin or support, while at leastone contaminant or impurity flows through. Binding mode may be used, forinstance, in hydroxyapatite chromatography and ion exchangechromatography.

A “contaminant” refers to any foreign or objectionable molecule,particularly a biological macromolecule such as a DNA, an RNA, or aprotein, other than the protein being purified that is present in asample of a protein being purified. Contaminants include, for example,other host cell proteins from cells used to recombinantly express theprotein being purified, proteins that are part of an absorbent used inan affinity chromatography step that may leach into a sample duringprior affinity chromatography step, such as Protein A, and mis-foldedvariants of the target protein itself.

“Host cell proteins” include proteins encoded by the naturally-occurringgenome of a host cell into which DNA encoding a protein that is to bepurified is introduced. Host cell proteins may be contaminants of theprotein to be purified, the levels of which may be reduced bypurification. Host cell proteins can be assayed for by any appropriatemethod including gel electrophoresis and staining and/or ELISA assay,among others. Host cell proteins include, for example, Chinese HamsterOvary (CHO) proteins (CHOP) produced as a product of expression ofrecombinant proteins.

The term “high molecular weight aggregates” or “HMWA” refers to anassociation of at least two antibody molecules. The association mayarise by any method including, but not limited to, covalent,non-covalent, disulfide, or nonreducible crosslinking. The at least twomolecules may bind to the same or different antigens.

As used herein, the term “Protein A” and associated phrases, such as“Protein A-based support” are intended to include Protein A (e.g.,recombinant or isolated Protein A), or a functional variant thereof. Inone embodiment, the Protein A is full length Staphylococcal Protein A(SpA) composed of five domains of about 50-60 amino acid residues knownas E, D, A, B and C domains in order from the N-terminus. (Sjodhal Eur JBiochem 78: 471-490 (1977); Uhlen et al. J. Biol. Chem. 259: 1695-1702(1984)). These domains contain approximately 58 residues, each sharingabout 65%-90% amino acid sequence identity. Binding studies betweenProtein A and antibodies have shown that while all five domains of SpA(E, D, A, B and C) bind to an IgG via its Fc region, domains D and Eexhibit significant Fab binding (Ljungberg et al. Mol. Immunol.30(14):1279-1285 (1993); Roben et al. J. Immunol. 154:6437-6445 (1995);Starovasnik et al. Protein Sci 8:1423-1431 (1999). The Z-domain, afunctional analog and energy-minimized version of the B domain (Nilssonet al. Protein Eng 1:107-113 (1987)) was shown to have negligiblebinding to the antibody variable domain region (Cedergren et al. ProteinEng 6(4):441-448 (1993); Ljungberg et al. (1993) supra; Starovasnik etal. (1999) supra). Protein A can include the amino acid sequence of SpA(SEQ ID NO:11) shown in FIG. 4A, or an amino acid sequence substantiallyidentical thereto. In other embodiments, the Protein A is a functionalvariant of SpA that includes at least one domain chosen from E, D, A, Band/or C, or a modified form thereof. For example, the functionalvariant of SpA can include at least domain B of SpA, or a variant ofdomain B, having one or more substituted asparagine residues, alsoreferred to herein as domain Z. In one embodiment, the functionalvariant of SpA includes the amino acid sequence of SEQ ID NO:12) shownin FIG. 4B, or an amino acid sequence substantially identical thereto.Other permutations of functional variants of Protein A can be usedcomprising domain B, or a variant domain B, and one or more of: domainsA and/or C; domains E, A and/or C; or domains E, D, A and/or C. Anycombination of E, D, A, B and/or C, or a functional variant thereof, canbe used as long as the combination is capable of binding to the SDABmolecule.

“Ceramic hydroxyapatite” or ‘cHA” refers to an insoluble hydroxylatedcalcium phosphate, e.g., having the formula [CaO(PO₄)₆(OH)₂ orCa₁₀(PO₄)₆(OH)₂], which has been sintered at high temperatures into aspherical, macroporous ceramic form. The term “cHA” encompasses, but isnot limited to, Type I and Type II ceramic hydroxyapatite. Unlessspecified, “cHA” refers to any particle size: including, but not limitedto, 20, 40, and 80 μm.

To “purify” a polypeptide means to reduce the amounts of foreign orobjectionable elements, especially biological macromolecules such asproteins or DNA, that may be present in a sample of the protein. Thepresence of foreign proteins may be assayed by any appropriate methodincluding gel electrophoresis and staining and/or ELISA assay. Thepresence of DNA may be assayed by any appropriate method including gelelectrophoresis and staining and/or assays employing polymerase chainreaction. In embodiments, the polypeptide, e.g., the SDAB molecule, ispurified to at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher purity.A polypeptide is “separated” (or “removed”) from a mixture comprisingthe protein and other contaminants when the mixture is subjected to aprocess such that the concentration of the target polypeptide is higherin the resulting product than the starting product.

The methods and compositions of the present invention encompasspolypeptides having the sequences specified, or sequences substantiallyidentical or similar thereto, e.g., sequences at least 85%, 90%, 95%identical or higher to the sequence specified. In the context of anamino acid sequence, the term “substantially identical” is used hereinto refer to a first amino acid that contains a sufficient or minimumnumber of amino acid residues that are i) identical to, or ii)conservative substitutions of aligned amino acid residues in a secondamino acid sequence such that the first and second amino acid sequencescan have a common structural domain and/or common functional activity.For example, amino acid sequences containing a common structural domainhaving at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identity to a reference sequence.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” when referring to proteins of the present invention include anypolypeptides which retain at least some of the functional properties ofthe corresponding native antibody or polypeptide. Fragments ofpolypeptides of the present invention include proteolytic fragments, aswell as deletion fragments, in addition to specific antibody fragmentsdiscussed elsewhere herein. Variants of the polypeptides of the presentinvention include fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally or benon-naturally occurring. Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of the fragments of the presentinvention are polypeptides which have been altered so as to exhibitadditional features not found on the native polypeptide. Examplesinclude fusion proteins. Variant polypeptides may also be referred toherein as “polypeptide analogs.” As used herein a “derivative” of apolypeptide refers to a subject polypeptide having one or more residueschemically derivatized by reaction of a functional side group. Alsoincluded as “derivatives” are those polypeptides which contain one ormore naturally occurring amino acid derivatives of the twenty standardamino acids. For example, 4-hydroxyproline may be substituted forproline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.

The term “functional variant” refers polypeptides that have asubstantially identical amino acid sequence to the naturally-occurringsequence, or are encoded by a substantially identical nucleotidesequence, and are capable of having one or more activities of thenaturally-occurring sequence.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In a preferred embodiment, thelength of a reference sequence aligned for comparison purposes is atleast 30%, preferably at least 40%, more preferably at least 50%, 60%,and even more preferably at least 70%, 80%, 90%, 100% of the length ofthe reference sequence. The amino acid residues at corresponding aminoacid positions are then compared. When a position in the first sequenceis occupied by the same amino acid residue as the corresponding positionin the second sequence, then the molecules are identical at thatposition (as used herein amino acid or nucleic acid “identity” isequivalent to amino acid or nucleic acid “homology”).

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused unless otherwise specified) are a Blossum 62 scoring matrix with agap penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller ((1989)CABIOS, 4:11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid (SEQ ID NO:1) molecules of the invention. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to a protein (SEQ ID NO:1)protein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST can be utilized as described inAltschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizingBLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

Various aspects of the invention are described in further detail below.

Single Domain Antigen Binding (SDAB) Molecules

In certain embodiments, the SDAB molecules purified by the methods ofthe invention are single chain fusion polypeptides comprised of one ormore nanobody molecules. For example, the SDAB molecule can be a singlechain fusion polypeptide comprising one or more nanobody molecules, thatbinds to one or more target antigens connected via a linker, e.g., apeptide linker.

As used herein, a “fusion polypeptide” refers to a protein containingtwo or more operably associated, e.g., linked, moieties, e.g., proteinmoieties. Typically, the moieties are covalently associated. Themoieties can be directly associated, or connected via a spacer or linker(e.g., a linking group as described herein). A fusion polypeptide can beproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, e.g., byemploying blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers that give rise tocomplementary overhangs between two consecutive gene fragments that cansubsequently be annealed and reamplified to generate a chimeric genesequence (see, for example, Ausubel et al. (eds.) Current Protocols inMolecular Biology, John Wiley & Sons, 1992). Moreover, many expressionvectors are commercially available that encode a fusion moiety. In someembodiments, fusion polypeptides exist as oligomers, such as dimers ortrimers of a single contiguous polypeptides, or two or morenon-contiguous polypeptides. In other embodiments, additional amino acidsequences can be added to the N- or C-terminus of the fusion protein tofacilitate expression, steric flexibility, detection and/or isolation orpurification.

Single domain antigen binding (SDAB) molecules include molecules whosecomplementary determining regions are part of a single domainpolypeptide. Examples include, but are not limited to, heavy chainvariable domains, binding molecules naturally devoid of light chains,single domains derived from conventional 4-chain antibodies, engineereddomains and single domain scaffolds other than those derived fromantibodies. SDAB molecules may be any of the art, or any future singledomain molecules. SDAB molecules may be derived from any speciesincluding, but not limited to mouse, human, camel, llama, fish, shark,goat, rabbit, and bovine. This term also includes naturally occurringsingle domain antibody molecules from species other that Camelidae andsharks.

In one aspect of the invention, an SDAB molecule can be derived from avariable region of the immunoglobulin found in fish, such as, forexample, that which is derived from the immunoglobulin isotype known asNovel Antigen Receptor (NAR) found in the serum of shark. Methods ofproducing single domain molecules derived from a variable region of NAR(“IgNARs”) are described in WO 03/014161 and Streltsov (2005) ProteinSci. 14:2901-2909.

According to another aspect of the invention, an SDAB molecule is anaturally occurring single domain antigen binding molecule known asheavy chain devoid of light chains. Such single domain molecules aredisclosed in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature363:446-448, for example. For clarity reasons, this variable domainderived from a heavy chain molecule naturally devoid of light chain isknown herein as a VHH or nanobody to distinguish it from theconventional VH of four chain immunoglobulins. Such a VHH molecule canbe derived from Camelidae species, for example in camel, llama,dromedary, alpaca and guanaco. Other species besides Camelidae mayproduce heavy chain molecules naturally devoid of light chain; such VHHsare within the scope of the invention.

In certain embodiments, the SDAB molecule includes at least oneimmunoglobulin variable domain (including one, two and/or threecomplementarity determining regions (CDRs)), in the absence of acomplementary antibody variable chain (e.g., a heavy chain variableregion (VH) in the absence of the corresponding light chain variableregion (VL)), and/or an immunoglobulin constant region, e.g., an Fcregion (or a constant region or a portion thereof capable of binding toProtein A).

In certain embodiments, an SDAB molecule does not include antibodymolecules having a heavy and light antibody variable domains or chains(e.g., full length antibodies), or antigen-binding fragments thereofhaving heavy and light antibody fragments (e.g., Fab, F(ab′)₂ fragment,scFv having a light and heavy chain variable regions in a singlepolypeptide chain, or a Fv fragment consisting of the VL and VH domainsof a single arm of an antibody).

The SDAB molecules can be recombinant, CDR-grafted, humanized,camelized, de-immunized and/or in vitro generated (e.g., selected byphage display), as described in more detail below.

The term “antigen-binding” is intended to include the part of apolypeptide, e.g., a single domain molecule described herein, thatcomprises determinants that form an interface that binds to a targetantigen, or an epitope thereof. With respect to proteins (or proteinmimetics), the antigen-binding site typically includes one or more loops(of at least four amino acids or amino acid mimics) that form aninterface that binds to the target antigen. Typically, theantigen-binding site of the polypeptide, e.g., the single domainantibody molecule, includes at least one or two CDRs, or more typicallyat least three, four, five or six CDRs.

The VH and VL regions can be subdivided into regions ofhypervariability, termed “complementarity determining regions” (CDR),interspersed with regions that are more conserved, termed “frameworkregions” (FR). The extent of the framework region and CDRs has beenprecisely defined by a number of methods (see, Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and theAbM definition used by Oxford Molecular's AbM antibody modellingsoftware. See, generally, e.g., Protein Sequence and Structure Analysisof Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.:Duebel, S, and Kontermann, R., Springer-Verlag, Heidelberg). Generally,unless specifically indicated, the following definitions are used: AbMdefinition of CDR1 of the heavy chain variable domain and Kabatdefinitions for the other CDRs. In addition, embodiments of theinvention described with respect to Kabat or AbM CDRs may also beimplemented using Chothia hypervariable loops. Each VH and VL typicallyincludes three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “immunoglobulin variable domain” is frequently understood inthe art as being identical or substantially identical to a VL or a VHdomain of human or animal origin. It shall be recognized thatimmunoglobulin variable domain may have evolved in certain species,e.g., sharks and llama, to differ in amino acid sequence from human ormammalian VL or VH. However, these domains are primarily involved inantigen binding. The term “immunoglobulin variable domain” typicallyincludes at least one or two CDRs, or more typically at least threeCDRs.

A “constant immunoglobulin domain” or “constant region” is intended toinclude an immunoglobulin domain that is identical to or substantiallysimilar to a CL, CH1, CH2, CH3, or CH4, domain of human or animalorigin. See e.g. Charles A Hasemann and J. Donald Capra,Immunoglobulins: Structure and Function, in William E. Paul, ed.,Fundamental Immunology, Second Edition, 209, 210-218 (1989). The term“Fc region” refers to the Fc portion of the constant immunoglobulindomain that includes immunoglobulin domains CH2 and CH3 orimmunoglobulin domains substantially similar to these.

In certain embodiments, the SDAB molecule is a monovalent, or amultispecific molecule (e.g., a bivalent, trivalent, or tetravalentmolecule). In other embodiments, the SDAB molecule is a monospecific,bispecific, trispecific or tetraspecific molecule. Whether a molecule is“monospecific” or “multispecific,” e.g., “bispecific,” refers to thenumber of different epitopes with which a binding polypeptide reacts.Multispecific molecules may be specific for different epitopes of atarget polypeptide described herein or may be specific for a targetpolypeptide as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material.

As used herein the term “valency” refers to the number of potentialbinding domains, e.g., antigen binding domains, present in an SDABmolecule. Each binding domain specifically binds one epitope. When anSDAB molecule comprises more than one binding domain, each bindingdomain may specifically bind the same epitope, for an antibody with twobinding domains, termed “bivalent monospecific,” or to differentepitopes, for an SDAB molecule with two binding domains, termed“bivalent bispecific.” An SDAB molecule may also be bispecific andbivalent for each specificity (termed “bispecific tetravalentmolecules”). Bispecific bivalent molecules, and methods of making them,are described, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, thedisclosures of all of which are incorporated by reference herein.Bispecific tetravalent molecules, and methods of making them aredescribed, for instance, in WO 02/096948 and WO 00/44788, thedisclosures of both of which are incorporated by reference herein. Seegenerally, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos.4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al.,J. Immunol. 148:1547-1553 (1992).

In certain embodiments, the SDAB molecule is a single chain fusionpolypeptide comprising one or more single domain molecules (e.g.,nanobodies), devoid of a complementary variable domain or animmunoglobulin constant, e.g., Fc, region, that binds to one or moretarget antigens. An exemplary target antigen recognized by theantigen-binding polypeptides includes tumor necrosis factor α (TNF α).In certain embodiments, the antigen-binding single domain molecule bindsto a serum protein, e.g., a human serum proteins chosen from one or moreof serum albumin (human serum albumin (HSA)) or transferin.

TNFα

Tumor necrosis factor alpha is known in the art to the associated withinflammatory disorders such as rheumatoid arthritis, Crohn's disease,ulcerative colitis and multiple sclerosis. Both and the receptors(CD120a and CD120b) have been studied in great detail. TNFa TNFa in itsbioactive form is a trimer. Several strategies to antagonize the actionof TNFa using anti-TNFa antibodies have been developed and are currentlycommercially available, such as Remicade® and Humira®. Antibodymolecules against TNFa are known. Numerous examples of TNFa-bindingsingle domain antigen binding molecules (e.g., nanobodies) are disclosedin WO 2004/041862, WO 2004/041865, WO 2006/122786, the contents of allof which are incorporated by reference herein in their entirety.Additional examples of single domain antigen binding molecules aredisclosed in US 2006/286066, US 2008/0260757, WO 06/003388, US05/0271663, US 06/0106203, the contents of all of which are incorporatedby reference herein in their entirety. In other embodiments, mono-, bi-,tri- and other multi-specific single domain antibodies against TNFa anda serum protein, e.g., HSA, are disclosed in these references.

In specific embodiments, the TNFα-binding nanobody molecule comprisesone or more of the nanobodies disclosed in WO 2006/122786. For example,the TNFα-binding nanobody molecule can be a monovalent, bivalent,trivalent TNFα-binding nanobody molecule disclosed in WO 2006/122786.Exemplary TNFα-binding nanobodies include, but are not limited to, TNF1,TNF2, TNF3, humanized forms thereof (e.g., TNF29, TNF30, TNF31, TNF32,TNF33). Additional examples of monovalent TNFα-binding nanobodies aredisclosed in Table 8 of WO 2006/122786. Exemplary bivalent TNFα-bindingnanobody molecules include, but are not limited to, TNF55 and TNF56,which comprise two TNF30 nanobodies linked via a peptide linker to forma single fusion polypeptide (disclosed in WO 2006/122786). Additionalexamples of bivalent TNFα-binding nanobody molecules are disclosed inTable 19 of WO 2006/122786 as TNF4, TNF5, TNF6, TNF7, TNF8).

In other embodiments, the HSA-binding nanobody molecule comprises one ormore of the nanobodies disclosed in WO 2006/122786. For example, theHSA-binding nanobody molecule can be a monovalent, bivalent, trivalentHSA-binding nanobody molecule disclosed in WO 2006/122786. In otherembodiments, the HSA-binding nanobody molecule can be a monospecific ora multispecific molecule having at least one of the bindingspecificities bind to HSA. Exemplary TNFα-binding nanobodies include,but are not limited to, ALB1, humanized forms thereof (e.g., ALB6, ALB7,ALB8, ALB9, ALB10), disclosed in WO 06/122786.

In other embodiments, two or more of the single domain molecules of thenanobody molecules are fused, with or without a linking group, as agenetic or a polypeptide fusion. The linking group can be any linkinggroup apparent to those of skill in the art. For instance, the linkinggroup can be a biocompatible polymer with a length of 1 to 100 atoms. Inone embodiment, the linking group includes or consists of polyglycine,polyserine, polylysine, polyglutamate, polyisoleucine, or polyarginineresidues, or a combination thereof. For example, the polyglycine orpolyserine linkers can include at least five, seven eight, nine, ten,twelve, fifteen, twenty, thirty, thirty-five and forty glycine andserine residues. Exemplary linkers that can be used include Gly-Serrepeats, for example, (Gly)₄-Ser repeats of at one, two, three, four,five, six, seven or more repeats (SEQ ID NO:8). In embodiments, thelinker has the following sequences: (Gly)₄-Ser-(Gly)₃-Ser (SEQ ID NO:9)or ((Gly)₄-Ser)n, where n is 4, 5, or 6 (SEQ ID NO:10).

In one exemplary embodiment, an antigen-binding polypeptide composed ofa single chain polypeptide fusion of two single domain antibodymolecules (e.g., two camelid variable regions) that bind to a targetantigen, e.g., tumor necrosis factor (TNFαa), and one single domainantibody molecule (e.g., a camelid variable region) that binds to aserum protein, e.g., HSA, referred to herein as “ATN-103,” was shown tobind to Protein A, or a functional variant thereof. ATN-103 is ahumanized, trivalent, bispecific, TNFa-inhibiting fusion protein. Theantigen for this protein is tumor necrosis factor-alpha (TNF). FIG. 1provides a schematic representation of the predicted structure ofATN-103. This fusion protein is derived from camelids and has a highdegree of sequence and structural homology to human immunoglobulin VHdomains. Its single polypeptide chain is composed of two binding domainsto TNFα and one to human serum albumin (HSA), with two nine amino acidG-S linkers connecting the domains. A detailed description of ATN-103 isprovided in WO 06/122786.

The complete amino acid sequence of the ATN-103 polypeptide chainpredicted from the DNA sequence of the corresponding expression vectoris shown in FIG. 2 (SEQ ID NO:1) (residues are numbered starting withthe NH₂-terminus as Residue Number 1). The last amino acid residueencoded by the DNA sequence is S³⁶³ and constitutes the COOH-terminus ofthe protein. The predicted molecular mass for disulfide-bonded ATN-103(with no posttranslational modifications) is 38434.7 Da. ATN-103contains no N-linked glycosylation consensus sequence. The molecularmass observed for the predominant isoform by nanoelectrospray ionizationquadrupole time-of-flight mass spectrometry corresponds to 38433.9 Daconfirming the absence of post-translational modifications.

In FIG. 2, complementarity determining regions (CDR) are underlined (SEQID NOs:2-7). The predicted intramolecular disulfide bonds areillustrated by connections of the cysteine residues involved. Thebinding domains to TNF are shown in bold and the binding domain to HSAis shown in bold italics. The amino acid linkers connecting thesebinding domains are in italics. The signal peptide (⁻¹⁹MGW . . . VHS⁻¹)is also shown for the polypeptide chain.

Preparation of SDAB Molecules

The SDAB molecules may comprised of one or more single domain molecules(e.g., nanobodies) that are recombinant, CDR-grafted, humanized,camelized, de-immunized, and/or in vitro generated (e.g., selected byphage display). Techniques for generating antibodies and SDAB molecules,and modifying them recombinantly are known in the art and are describedin detail below.

Numerous methods known to those skilled in the art are available forobtaining antibodies. For example, monoclonal antibodies may be producedby generation of hybridomas in accordance with known methods. Hybridomasformed in this manner are then screened using standard methods, such asenzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance(BIACORE™) analysis, to identify one or more hybridomas that produce annanobody that specifically binds with a specified antigen. Any form ofthe specified antigen may be used as the immunogen, e.g., recombinantantigen, naturally occurring forms, any variants or fragments thereof,as well as antigenic peptide thereof.

One exemplary method of making antibodies and SDAB molecules includesscreening protein expression libraries, e.g., phage or ribosome displaylibraries. Phage display is described, for example, in Ladner et al.,U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; and WO 90/02809.

In addition to the use of display libraries, the specified antigen canbe used to immunize a non-human animal, e.g., a rodent, e.g., a mouse,hamster, or rat. In one embodiment, the non-human animal includes atleast a part of a human immunoglobulin gene. For example, it is possibleto engineer mouse strains deficient in mouse antibody production withlarge fragments of the human Ig loci. Using the hybridoma technology,antigen-specific monoclonal antibodies derived from the genes with thedesired specificity may be produced and selected. See, e.g., XENOMOUSE™,Green et al. (1994) Nature Genetics 7:13-21, US 2003-0070185, WO96/34096, published Oct. 31, 1996, and PCT Application No.PCT/US96/05928, filed Apr. 29, 1996.

In another embodiment, an SDAB molecule is obtained from the non-humananimal, and then modified, e.g., humanized, deimmunized, chimeric, maybe produced using recombinant DNA techniques known in the art. A varietyof approaches for making chimeric antibodies and SDAB molecules havebeen described. See e.g., Morrison et al., Proc. Natl. Acad. Sci. U.S.A.81:6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., U.S.Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi etal., European Patent Publication EP171496; European Patent Publication0173494, United Kingdom Patent GB 2177096B. Humanized antibodies andSDAB molecules may also be produced, for example, using transgenic micethat express human heavy and light chain genes, but are incapable ofexpressing the endogenous mouse immunoglobulin heavy and light chaingenes. Winter describes an exemplary CDR-grafting method that may beused to prepare the humanized antibodies and SDAB molecule describedherein (U.S. Pat. No. 5,225,539). All of the CDRs of a particular humanantibody may be replaced with at least a portion of a non-human CDR, oronly some of the CDRs may be replaced with non-human CDRs. It is onlynecessary to replace the number of CDRs required for binding of thehumanized antibody and SDAB molecule to a predetermined antigen.

Humanized antibodies can be generated by replacing sequences of the Fvvariable domain that are not directly involved in antigen binding withequivalent sequences from human Fv variable domains. Exemplary methodsfor generating humanized antibodies or fragments thereof are provided byMorrison (1985) Science 229:1202-1207; by Oi et al. (1986) BioTechniques4:214; and by U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S.Pat. No. 5,693,762; U.S. Pat. No. 5,859,205; and U.S. Pat. No.6,407,213. Those methods include isolating, manipulating, and expressingthe nucleic acid sequences that encode all or part of immunoglobulin Fvvariable domains from at least one of a heavy or light chain. Suchnucleic acids may be obtained from a hybridoma producing an nanobodyagainst a predetermined target, as described above, as well as fromother sources. The recombinant DNA encoding the humanized SDAB molecule,e.g., nanobody molecule, can then be cloned into an appropriateexpression vector.

In certain embodiments, a humanized SDAB molecule, e.g., nanobodymolecule, is optimized by the introduction of conservativesubstitutions, consensus sequence substitutions, germline substitutionsand/or backmutations. Such altered immunoglobulin molecules can be madeby any of several techniques known in the art, (e.g., Teng et al., Proc.Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983; Kozbor et al., ImmunologyToday, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982),and may be made according to the teachings of PCT Publication WO92/06193or EP 0239400).

Techniques for humanizing SDAB molecules, e.g., nanobody molecules, aredisclosed in WO 06/122786.

An SDAB molecule, e.g., nanobody molecule, may also be modified byspecific deletion of human T cell epitopes or “deimmunization” by themethods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy andlight chain variable domains of, e.g., a nanobody can be analyzed forpeptides that bind to MHC Class II; these peptides represent potentialT-cell epitopes (as defined in WO 98/52976 and WO 00/34317). Fordetection of potential T-cell epitopes, a computer modeling approachtermed “peptide threading” can be applied, and in addition a database ofhuman MHC class II binding peptides can be searched for motifs presentin the V_(H) and V_(L) sequences, as described in WO 98/52976 and WO00/34317. These motifs bind to any of the 18 major MHC class II DRallotypes, and thus constitute potential T cell epitopes. PotentialT-cell epitopes detected can be eliminated by substituting small numbersof amino acid residues in the variable domains, or preferably, by singleamino acid substitutions. Typically, conservative substitutions aremade. Often, but not exclusively, an amino acid common to a position inhuman germline antibody sequences may be used. Human germline sequences,e.g., are disclosed in Tomlinson, et al. (1992) J. Mol. Biol.227:776-798; Cook, G. P. et al. (1995) Immunol. Today Vol. 16 (5):237-242; Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; andTomlinson et al. (1995) EMBO J. 14:4628-4638. The V BASE directoryprovides a comprehensive directory of human immunoglobulin variableregion sequences (compiled by Tomlinson, I. A. et al. MRC Centre forProtein Engineering, Cambridge, UK). These sequences can be used as asource of human sequence, e.g., for framework regions and CDRs.Consensus human framework regions can also be used, e.g., as describedin U.S. Pat. No. 6,300,064.

The SDAB molecules, e.g., nanobody molecules, can be produced by livinghost cells that have been genetically engineered to produce the protein.Methods of genetically engineering cells to produce proteins are wellknown in the art. See e.g. Ausabel et al., eds. (1990), CurrentProtocols in Molecular Biology (Wiley, New York). Such methods includeintroducing nucleic acids that encode and allow expression of theprotein into living host cells. These host cells can be bacterial cells,fungal cells, or, preferably, animal cells grown in culture. Bacterialhost cells include, but are not limited to, Escherichia coli cells.Examples of suitable E. coli strains include: HB101, DH5a, GM2929,JM109, KW251, NM538, NM539, and any E. coli strain that fails to cleaveforeign DNA. Fungal host cells that can be used include, but are notlimited to, Saccharomyces cerevisiae, Pichia pastoris and Aspergilluscells. A few examples of animal cell lines that can be used are CHO,VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and WI38. New animal cell linescan be established using methods well know by those skilled in the art(e.g., by transformation, viral infection, and/or selection).Optionally, the protein can be secreted by the host cells into themedium.

Modified SDAB Molecules

The SDAB molecule, e.g., nanobody molecule, purified using the methodsof the invention can have an amino acid sequence that differs at leastone amino acid position in one of the framework regions from the aminoacid sequence of a naturally occurring domain, e.g., VH domain.

It shall be understood that the amino acid sequences of the some of theSDAB molecules of the invention, such as the humanized SDAB molecules,can differ at least one amino acid position in at least one of theframework regions from the amino acid sequences of naturally occurringdomain, e.g., a naturally occurring VHI-I domains.

The invention also includes methods of purifying derivatives of the SDABmolecules. Such derivatives can generally be obtained by modification,and in particular by chemical and/or biological (e.g. enzymatical)modification, of the SDAB molecules and/or of one or more of the aminoacid residues that form the SDAB molecules disclosed herein.

Examples of such modifications, as well as examples of amino acidresidues within the SDAB molecule sequence that can be modified in sucha manner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the SDAB molecule,and in particular of one or more functional groups, residues or moietiesthat confer one or more desired properties or functionalities to theSDAB molecules. Example of such functional groups will be clear to theskilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that that increase the half-life, the solubilityand/or the absorption of the SDAB molecule, that reduce theimmunogenicity and/or the toxicity of the SDAB molecule, that eliminateor attenuate any undesirable side effects of the SDAB molecule, and/orthat confer other advantageous properties to and/or reduce the undesiredproperties of the SDAB molecule; or any combination of two or more ofthe foregoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and—148-single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One widely used techniques for increasing the half-life and/or thereducing immunogenicity of pharmaceutical proteins comprises attachmentof a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in an SDAB molecule, an SDABmolecule may be modified so as to suitably introduce one or morecysteine residues for attachment of PEG, or an amino acid sequencecomprising one or more cysteine residues for attachment of PEG may befused to the N- and/or C-terminus of a Nanobody of the invention, allusing techniques of protein engineering known per se to the skilledperson.

Preferably, for the SDAB molecule, a PEG is used with a molecular weightof more than 5000, such as more than 10,000 and less than 200,000, suchas less than 100,000; for example in the range of 20,000-80,000.

With regard to pegylation, its should be noted that generally, theinvention also encompasses any SDAB molecule that has been pegylated atone or more amino acid positions, preferably in such a way that saidpegylation either (1) increases the half-life in vivo; (2) reducesimmunogenicity; (3) provides one or more further beneficial propertiesknown per se for pegylation; (4) does not essentially affect theaffinity of the SDAB molecule (e.g. does not reduce said affinity bymore than 90%, preferably not by more than 50%, and by no more than 10%,as determined by a suitable assay, such as those described in theExamples below); and/or (4) does not affect any of the other desiredproperties of the SDAB molecule. Suitable PEG-groups and methods forattaching them, either specifically or non-specifically, will be clearto the skilled person.

Suitable kits and reagents for such pegylation can for example beobtained from Nektar (CA, USA).

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the SDAB molecule.

Chromatographic Processes

The process of purifying a protein often requires numerous steps, witheach step resulting in a further reduction in yield. Protein A-basedchromatography is one of many techniques commonly used. Proteinpurification by Protein A-based chromatography may be performed in acolumn containing an immobilized Protein A ligand (typically a columnpacked with modified support of methacrylate copolymer or agarose beadsto which is affixed an adsorbent consisting of Protein A or functionalderivatives thereof). The column is typically equilibrated with a bufferat high salt concentration and a sample containing a mixture of proteins(the target protein, plus contaminating proteins) in a compatiblenon-denaturing high salt solution, is loaded onto the column. As themixture passes through the column, the target protein binds to theadsorbent within the column, while unbound contaminants flow through.Bound protein is then eluted from the column with a reduced saltconcentration. Typically, the target protein may be recovered by elutingthe column with a salt concentration applied in a gradual or step-wisereduced gradient, to selectively release the various bound proteins atthe particular salt concentration conducive to their release, andcollecting discreet fractions until the fraction containing the morepurified protein is obtained. By collecting flow-through fractions overdiscreet periods of time, fractions containing specific proteins can beisolated. In a process where a target protein is bound to the column(while allowing contaminants to flow through), adsorbents having greateraffinity to Protein A are typically used to bind a broader range ofproteins which will be collected in a specific fraction conducive to therelease of the protein.

The process of the invention can be used in combination with otherprotein purification methodologies, such as salt precipitation, affinitychromatography, hydroxyapatite chromatography, reverse phase liquidchromatography, ion-exchange chromatography, or any other commonly usedprotein purification technique. It is contemplated, however, that theprocess of the present invention will eliminate or significantly reducethe need for other purification steps.

Any or all chromatographic steps of the present invention can be carriedout by any mechanical means. Chromatography may be carried out, forexample, in a column. The column may be run with or without pressure andfrom top to bottom or bottom to top. The direction of the flow of fluidin the column may be reversed during the chromatography process.Chromatography may also be carried out using a batch process in whichthe solid media is separated from the liquid used to load, wash, andelute the sample by any suitable means, including gravity,centrifugation, or filtration. Chromatography may also be carried out bycontacting the sample with a filter that absorbs or retains somemolecules in the sample more strongly than others. In the followingdescription, the various embodiments of the present invention aredescribed in the context of chromatography carried out in a column. Itis understood, however, that use of a column is merely one of severalchromatographic modalities that may be used, and the illustration of thepresent invention using a column does not limit the application of thepresent invention to column chromatography, as those skilled in the artmay readily apply the teachings to other modalities as well, such asthose using a batch process or filter.

Suitable supports may be any currently available or later developedmaterials having the characteristics necessary to practice the claimedmethod, and may be based on any synthetic, organic, or natural polymers.For example, commonly used support substances include organic materialssuch as cellulose, polystyrene, agarose, sepharose, polyacrylamidepolymethacrylate, dextran and starch, and inorganic materials, such ascharcoal, silica (glass beads or sand) and ceramic materials. Suitablesolid supports are disclosed, for example, in Zaborsky “ImmobilizedEnzymes” CRC Press, 1973, Table IV on pages 28-46.

Prior to equilibration and chromatography, the chromatography media (thesupport and adsorbent affixed to the support) may be pre-equilibrated ina chosen solution, e.g. a salt and/or buffer solution. Pre-equilibrationserves the function of displacing a solution used for regeneratingand/or storing the chromatography medium. One of skill in the art willrealize that the composition of the pre-equilibration solution dependson the composition of the storage solution and the solution to be usedfor the subsequent chromatography. Thus, appropriate pre-equilibrationsolutions may include the same buffer or salt used for performing thechromatography, optionally, at a higher concentration than is used toperform chromatography. Buffers and salts that can be used forchromatography are discussed below. For example, when the solution usedto perform chromatography comprises sodium phosphate at a givenconcentration, pre-equilibration may take place in a in a solutioncomprising sodium phosphate at a higher concentration. As anillustration of this, if the solution used to perform chromatographycomprises sodium phosphate at between about 0.5 millimolar and about 50millimolar, pre-equilibration may occur in a solution comprising sodiumphosphate at concentrations between about 0.2 molar and about 0.5 molar,more preferably in concentrations of sodium phosphate between about 0.3molar and about 0.4 molar, inclusive.

Before the sample is applied to the column, the column can beequilibrated in the buffer or salt that will be used to chromatographthe protein. As discussed below, chromatography (and loading of theprotein to be purified) can occur in a variety of buffers or saltsincluding sodium, potassium, ammonium, magnesium, calcium, chloride,fluoride, acetate, phosphate, and/or citrate salts and/or Tris buffer.Citrate buffers and salts are preferred by those skilled in the art fortheir ease of disposal. Such buffers or salts can have a pH of at leastabout 5.5. In some embodiments, equilibration may take place in asolution comprising a Tris or a sodium phosphate buffer. Optionally, thesodium phosphate buffer is at a concentration between about 0.5millimolar and about 50 millimolar, more preferably at a concentrationbetween about 15 millimolar and 35 millimolar. Preferably, equilibrationtakes place at a pH of at least about 5.5. Equilibration may take placeat pHs between about 6.0 and about 8.6, preferably at pHs between about6.5 and 7.5. Most preferably, the solution comprises a sodium phosphatebuffer at a concentration of about 25 millimolar and at a pH of about6.8.

Suitable buffers include, but are not limited to phosphate buffers, Trisbuffers, acetate buffers, and/or citrate buffers. Acceptable salts mayinclude, but are not limited to sodium chloride, ammonium chloride,potassium chloride, sodium acetate, ammonium acetate, sodium sulfate,ammonium sulfate, ammonium thiocyanate, sodium citrate, sodiumphosphate, and potassium, magnesium, and calcium salts thereof, andcombinations of these salts. In other embodiments, the salts includesodium citrate and sodium chloride. Acceptable ranges of saltconcentrations used for chromatographic systems are typically in therange of from 0 to about 2M sodium citrate, 0 to about 4M sodiumchloride, 0 to about 3M ammonium sulfate, 0 to about 1M sodium sulfateand 0 to about 2M sodium phosphate. The ranges of salt concentration mayinclude 0 to about 1M sodium citrate, 0 to about 2M sodium chloride, 0to about 1.5M ammonium sulfate, 0 to about 1M sodium sulfate and 0 toabout 1.5M sodium phosphate. Other buffers and salts can also be used.After loading, the adsorbent can be washed with more of the samesolution to cause the target protein (unbound to the adsorbent) to flowthrough the adsorbent. The protein is then collected in the flow-throughfraction. Conditions for binding contaminants, while the target proteindoes not bind, can be easily optimized by those skilled in the art. Thesalt concentrations discussed herein are exemplary, and other salts andsalt concentrations can be used by varying flow rates, temperatures, andelution times as is known in the art.

Conditions under which columns are used vary with the specific columnsas is known in the art. For most proteins of interest, the pH range maybe between about 6.0 and about 8.6, or alternatively between about 6.5and about 7.5. However, certain proteins are known to be resistant to pHextremes, and a broader range may be possible. Typical conditionsinclude a pH range of 5-7 and a sodium citrate concentration range of 0to about 0.8M (e.g. 0.5M sodium citrate, pH 6.0).

One skilled in the art will be guided by the knowledge in the art indetermining which buffer or salt is appropriate for the particularprotein being purified. Moreover, a skilled artisan can easily determinethe optimal concentration of the selected buffer or salt to use by, forexample, establishing particular buffer or salt conditions under whichcontaminants bind to a column while the protein of interest flowsthrough in the flow-through fraction. Fractions of the effluent of thecolumn can be collected and analyzed to determine the concentration ofbuffer or salt at which the target protein and the contaminants elute.Suitable analyses include, for example, a measurement of electricalconductance with a conductivity meter (to determine the saltconcentration in the sample) plus gel electrophoresis or ELISA assay (todetermine the identity of the proteins in the sample). Optionally, thecolumn can be washed with more of the same solution in which the proteinsample was loaded, and this wash solution can also be collected andcombined with the flow-through liquid.

Subsequent to collection of the flow through and, optionally, the wash,which comprises the protein being purified, proteins that may remainbound to the column may be released by stripping the chromatographymedium using a solution comprising the buffer or salt used forchromatography, but at a lower ionic strength to release the contaminantproteins. Then, the column may be regenerated using a solution that willhave the effect of releasing most or all proteins from thechromatography medium and reducing or eliminating any microbialcontamination that may be present in the chromatography medium. In oneembodiment, such a solution may comprise sodium hydroxide. Otherreagents can also be used. Subsequently, the column may be rinsed andstored in a solution that can discourage microbial growth. Such asolution may comprise sodium hydroxide, but other reagents can also beappropriate.

Protein concentration of a sample at any stage of purification can bedetermined by any suitable method. Such methods are well known in theart and include: 1) colorimetric methods such as the Lowry assay, theBradford assay, the Smith assay, and the colloidal gold assay; 2)methods utilizing the UV absorption properties of proteins; and 3)visual estimation based on stained protein bands on gels relying oncomparison with protein standards of known quantity on the same gel. Seee.g. Stoschek (1990), Quantitation of Protein, in Guide to ProteinPurification, Methods in Enzymol. 182: 50-68.

The target protein, as well as contaminating proteins that may bepresent in a sample, can be monitored by any appropriate means.Preferably, the technique should be sensitive enough to detectcontaminants in the range between about 2 parts per million (ppm)(calculated as nanograms per milligram of the protein being purified)and 500 ppm. For example, enzyme-linked immunosorbent assay (ELISA), amethod well known in the art, may be used to detect contamination of theprotein by the second protein. See e.g. Reen (1994), Enzyme-LinkedImmunosorbent Assay (ELISA), in Basic Protein and Peptide Protocols,Methods Mol. Biol. 32: 461-466, which is incorporated herein byreference in its entirety. In one aspect, contamination of the proteinby such other proteins can be reduced after the methods describedherein, preferably by at least about two-fold, more preferably by atleast about three-fold, more preferably by at least about five-fold,more preferably by at least about ten-fold, more preferably by at leastabout twenty-fold, more preferably by at least about thirty-fold, morepreferably by at least about forty-fold, more preferably by at leastabout fifty-fold, more preferably by at least about sixty-fold, morepreferably by at least about seventy-fold, more preferably by at leastabout 80-fold, more preferably by at least about 90-fold, and mostpreferably by at least about 100-fold.

In another aspect, contamination of the protein by such other proteinsafter the methods described herein is not more than about 10,000 ppm,preferably not more than about 2500 ppm, more preferably not more thanabout 400 ppm, more preferably not more than about 360 ppm, morepreferably not more than about 320 ppm, more preferably not more thanabout 280 ppm, more preferably not more than about 240 ppm, morepreferably not more than about 200 ppm, more preferably not more thanabout 160 ppm, more preferably not more than about 140 ppm, morepreferably not more than about 120 ppm, more preferably not more thanabout 100 ppm, more preferably not more than about 80 ppm, morepreferably not more than about 60 ppm, more preferably not more thanabout 40 ppm, more preferably not more than about 30 ppm, morepreferably not more than about 20 ppm, more preferably not more thanabout 10 ppm, and most preferably not more than about 5 ppm. Suchcontamination can range from undetectable levels to about 10 ppm or fromabout 10 ppm to about 10,000 ppm. If a protein is being purified forpharmacological use, one of skill in the art will realize that thepreferred level of the second protein can depend on the weekly dose ofthe protein to be administered per patient, with the aim that thepatient will not receive more than a certain amount of a contaminatingprotein per week. Thus, if the required weekly dose of the protein isdecreased, the level of contamination by a second protein may possiblyincrease.

The amount of DNA that may be present in a sample of the protein beingpurified can be determined by any suitable method. For example, one canuse an assay utilizing polymerase chain reaction. Optionally, thetechnique can detect DNA contamination at levels of 10 picograms permilligram of protein and greater. DNA levels can be reduced by HIC,optionally by about two-fold, preferably by about five-fold, morepreferably by about ten-fold, more preferably by about fifteen-fold,most preferably by about 20-fold. Optionally, levels of DNA afterhydroxyapatite chromatography are less than about 20 picograms permilligram of protein, preferably less than 15 picograms per milligram ofprotein, more preferably less than 10 picograms per milligram ofprotein, most preferably less than 5 picograms per milligram of protein.

Protein A-Based Chromatography

In one embodiment, the harvest media containing the antibodypreparations may be purified by Protein A chromatography. StaphylococcalProtein A (SpA) is a 42 kDa protein composed of five nearly homologousdomains named as E, D, A, B and C in order from the N-terminus (SjodhalEur J Biochem 78: 471-490 (1977); Uhlen et al. J. Biol. Chem. 259:1695-1702 (1984)). These domains contain approximately 58 residues, eachsharing about 65%-90% amino acid sequence identity. Binding studiesbetween Protein A and antibodies have shown that while all five domainsof SpA (E, D, A, B and C) bind to an IgG via its Fc region, domains Dand E exhibit significant Fab binding (Ljungberg et al. Mol. Immunol.30(14):1279-1285 (1993); Roben et al. J. Immunol. 154:6437-6445 (1995);Starovasnik et al. Protein Sci 8:1423-1431 (1999). The Z-domain, afunctional analog and energy-minimized version of the B domain (Nilssonet al. Protein Eng 1:107-113 (1987)) was shown to have negligiblebinding to the antibody variable domain region (Cedergren et al. ProteinEng 6(4):441-448 (1993); Ljungberg et al. (1993) supra; Starovasnik etal. (1999) supra).

Until recently, commercially available Protein A stationary phasesemployed SpA (isolated from Staphylococcus aureus or expressedrecombinantly) as their immobilized ligand. Using these columns, it hasnot been possible to use alkaline conditions for column regeneration andsanitation as is typically done with other modes of chromatography usingnon-proteinaceous ligands (Ghose et al. Biotechnology and BioengineeringVol. 92 (6) (2005)). A new resin (MabSELECT™ SuRe) has been developed towithstand stronger alkaline conditions (Ghose et al. (2005) supra).Using protein engineering techniques, a number of asparagine residueswere replaced in the Z-domain of protein A and a new ligand was createdas a tetramer of four identically modified Z-domains (Ghose et al.(2005) supra).

Accordingly, purification methods can be carried out using commerciallyavailable Protein A columns according to manufacturers' specification.As described in the appended examples, MabSELECT™ columns or MabSELECT™SuRe columns (GE Healthcare Products) can be used. MabSELECT™ is acommercially available resin containing recombinant SpA as itsimmobilized ligand. It captures antibody molecules from large media bypacked bed chromatography. The recombinant Protein A ligand ofMabSELECT™ is engineered to favor an orientation of the Protein A ligandthat enhances binding capacity for IgG. The specificity of Protein Aligand to the binding region of IgG is similar to that of native ProteinA. MabSELECT™ SuRe columns have a similar highly cross-linked agarosematrix used for MabSELECT™, the ligand used is a tetramer of fouridentically modified Z-domains (GE Healthcare Products). Othercommercially available sources of Protein A column include, but are notlimited to, PROSEP-ATM (Millipore, U.K.), which consists of Protein Acovalently coupled to controlled pore glass, can be usefully employed.Other useful Protein A formulations include Protein A Sepharose FASTFLOW™ (Amersham Biosciences, Piscataway, N.J.), and TOYOPEARL™ 650MProtein A (TosoHaas Co., Philadelphia, Pa.).

Hydroxyapatite Resins

Various hydroxyapatite chromatographic resins are availablecommercially, and any available form of the material can be used in thepractice of this invention. A detailed description of the conditionssuitable for hydroxyapatite chromatography is provided in WO 05/044856,the contents of which are incorporated by reference herein in itsentirety.

In one embodiment of the invention, the hydroxyapatite is in acrystalline form. Hydroxyapatites for use in this invention may be thosethat are agglomerated to form particles and sintered at hightemperatures into a stable porous ceramic mass. The particle size of thehydroxyapatite may vary widely, but a typical particle size ranges from1 μm to 1,000 μm in diameter, and may be from 10 μm to 100 μm. In oneembodiment of the invention, the particle size is 20 μm. In anotherembodiment of the invention, the particle size is 40 μm. In yet anotherembodiment of the invention, the particle size is 80 μm.

A number of chromatographic supports may be employed in the preparationof cHA columns, the most extensively used are Type I and Type Ihydroxyapatite. Type I has a high protein binding capacity and bettercapacity for acidic proteins. Type II, however, has a lower proteinbinding capacity, but: has better resolution of nucleic acids andcertain proteins. The Type II material also has a very low affinity foralbumin and is especially suitable for the purification of many speciesand classes of immunoglobulins. The choice of a particularhydroxyapatite type can be determined by the skilled artisan.

This invention may be used with a hydroxyapatite resin that is loose,packed in a column, or in a continuous annual chromatograph. In oneembodiment of the invention, ceramic hydroxyapatite resin is packed in acolumn. The choice of column dimensions can be determined by the skilledartisan. In one embodiment of the invention, a column diameter of atleast 0.5 cm with a bed height of about 20 cm may be used for smallscale purification.

In an additional embodiment of the invention, a column diameter of fromabout cm to about 60 cm may be used. In yet another embodiment of theinvention, a column diameter of from 60 cm to 85 cm may be used. Incertain embodiments of the invention, a slurry of ceramic hydroxyapatiteresin in 200 mM Na₂HPO4 solution at pH 9.0 may be used to pack thecolumn at a constant flow rate of about 4 cm/min or with gravity.

Buffer Compositions and Loading Conditions for Hydroxyapatite Resins

Before contacting the hydroxyapatite resin with the antibodypreparation, it may be necessary to adjust parameters such as pH, ionicstrength, and temperature and in some instances the addition ofsubstances of different kinds. Thus, it is an optional step to performan equilibration of the hydroxyapatite matrix by washing it with asolution (e.g., a buffer for adjusting pH, ionic strength, etc., or forthe introduction of a detergent) bringing the necessary characteristicsfor purification of the antibody preparation.

In combination binding/flow-through mode hydroxyapatite chromatography,the hydroxyapatite matrix is equilibrated and washed with a solution,thereby bringing the necessary characteristics for purification of theantibody preparation. In one embodiment of the invention, the matrix maybe equilibrated using a solution containing from 0.01 to 2.0 M NaCl atslightly basic to slightly acidic pH. For example, the equilibrationbuffer may contain 1 to 20 mM sodium phosphate, in another embodiment itmay contain 1 to 10 mM sodium phosphate, in another embodiment it maycontain 2 to 5 mM sodium phosphate, in another embodiment it may contain2 mM sodium phosphate, and in another embodiment may contain 5 mM sodiumphosphate. The equilibration buffer may contain 0.01 to 2.0 M NaCl, inone embodiment, 0.025 to 0.5 M NaCl, in another embodiment, 0.05 M NaCl,and in another embodiment, 0.1 M NaCl. The pH of the load buffer mayrange from 6.2 to 8.0. In one embodiment, the pH may be from 6.6 to 7.7,and in another embodiment the pH may be 7.3. The equilibration buffermay contain 0 to 200 mM arginine, in another embodiment it may contain120 mM arginine, and in another embodiment it may contain 100 mMarginine. The equilibration buffer may contain 0 to 200 mM HEPES, inanother embodiment it may contain 20 mM HEPES, and in another embodimentit may contain 100 mM HEPES.

The SDAB molecule preparation may also be buffer exchanged into anappropriate buffer or load buffer in preparation for flow-through modehydroxyapatite chromatography. In one embodiment of the invention, theantibody preparation may be buffer exchanged into a load buffercontaining 0.2 to 2.5 M NaCl at slightly acidic to slightly basic pH.For example, the load buffer may contain 1 to 20 mM sodium phosphate, inanother embodiment it may contain 2 to 8 mM sodium phosphate, in anotherembodiment it may contain 3 to 7 mM sodium phosphate, and in anotherembodiment may contain 5 mM sodium phosphate. The load buffer maycontain 0.2 to 2.5 M NaCl in one embodiment, 0.2 to 1.5 M NaCl, inanother embodiment, 0.3 to 1.0 M NaCl, and in another embodiment, 350 mMNaCl. The pH of the load buffer may range from 6.4 to 7.6. In oneembodiment, the pH may be from 6.5 to 7.0, and in another embodiment thepH may be 6. 8.

The contacting of an SDAB molecule preparation to the hydroxyapatiteresin in either binding mode, flow-through mode, or combinations thereofmay be performed in a packed bed column, a fluidized/expanded bed columncontaining the solid phase matrix, and/or in a simple batch operationwhere the solid phase matrix is mixed with the solution for a certaintime.

After contacting the hydroxyapatite resin with the antibody preparationthere is optionally performed a washing procedure. However, in somecases where very high purity of the immunoglobulin is not critical or:additional flow-through antibody is not required, the washing proceduremay be omitted, saving a process-step as well as washing solution. Thewashing buffers employed will depend on the nature of the hydroxyapatiteresin, the mode of hydroxyapatite chromatography being employed, andtherefore can be determined by one of ordinary skill in the art. Inflow-through mode and combination binding/flow-through mode, thepurified antibody flow-through obtained after an optional wash of thecolumn may be pooled with other purified antibody fractions.

In binding mode, the SDAB molecule may be eluted from the column afteran optional washing procedure. For elution of the antibody from thecolumn, this invention uses a high ionic strength phosphate buffercontaining about 0.2 to 2.5 M NaCl at slightly acidic to slightly basicpH. For example, the elusion buffer may contain 1 to 20 mM sodiumphosphate, in another embodiment it may contain 2 to 8 mM sodiumphosphate, in another embodiment it may contain 2 to 6 mM sodiumphosphate, in another embodiment may contain 3 mM sodium phosphate, andin another embodiment may contain 5 mM sodium phosphate. The elusionbuffer may contain 0.2 to 2.5 M NaCl, in one embodiment, 0.2 to 1.5 MNaCl, in another embodiment, 0.3 to 1.1 M NaCl, in another embodiment,1.0 M NaCl, and in another embodiment, 0.35 M NaCl. The pH of theelusion buffer may range from 6.4 to 7.6. In one embodiment, the pH maybe from 6.5 to 7.3, in another embodiment the pH may be 7.2, and inanother embodiment the pH may be 6.8. The elusion buffer may be alteredfor elusion of the antibody from the column in a continuous or stepwisegradient.

In both the binding mode, flow-through mode, and combinations thereof, asolid phase matrix may optionally be cleaned, i.e. stripped andregenerated, after elusion or flow through of the antibody. Thisprocedure is typically performed regularly to minimize the building upof impurities on the surface of the solid phase and/or to sterilize thematrix to avoid contamination of the product with microorganisms.

Buffer components may be adjusted according to the knowledge of theperson of ordinary skill in the art.

Additional Optional Steps

Although it has been discovered that hydroxyapatite chromatography canbe used alone to separate monomeric IgG from aggregates, as mentionedabove, the purification method of the invention can be used incombination with other protein purification techniques. In oneembodiment of the invention, one or more steps preceding thehydroxyapatite step may be desirable to reduce the load challenge of thecontaminants or impurities. In another embodiment of the invention, oneor more purification steps following the hydroxyapatite step may bedesirable to remove additional contaminants or impurities.

The cHA purification procedure described may optionally be i combinedwith other purification steps, including but not limited to, Protein Achromatography, affinity chromatography, hydrophobic interactionchromatography, immobilized metal affinity chromatography, sizeexclusion chromatography, diafiltration, ultrafiltration, viral removalfiltration, and/or ion exchange chromatography.

In one embodiment, prior to the cHA purification step, the harvest mediamay optionally be initially purified by a Protein A chromatography step.For example, PROSEP-ATM (Millipore, U.K.), which consists of Protein Acovalently coupled to controlled pore glass, can be usefully employed.Other useful Protein A formulations include Protein A Sepharose FASTFLOW™ (Amersham Biosciences, Piscataway, N.J.), TOYOPEARL™ 650M ProteinA (TosoHaas Co., Philadelphia, Pa.), and MABSELECT™ columns (AmershamBiosciences, Piscataway, N.J.).

As an optional step prior to the cHA purification, ion exchangechromatography may be employed. In this regard various anionic orcationic substituents may be attached to matrices in order to formanionic or cationic supports for chromatography. Anionic exchangesubstituents include diethylaminoethyl (DEAE), trimethylandinoethylacrylamide (TMAE), quaternary aminoethyl (QAE) and quaternary amine (Q)groups. Cationic exchange substituents include carboxymethyl (CM),sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S).Cellulosic ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32and CM-52 are available from Whatman Ltd. Maidstone, Kent, U.K.Sephadex-based and cross-linked; ion exchangers are also known. Forexample, DEAE-, QAE-, CM-, and SP Sephadex, and DEAE-, Q-, CM- andS-Sepharose, and Sepharose are all available from Amersham Biosciences,Piscataway, N.J. Further, both DEAE and CM derivitized ethyleneglycol-methacrylate copolymer such as TOYOPEARL™ DEAE-6505 or M andTOYOPEARL™ CM-650S or M are available from Toso Haas Co., Philadelphia,Pa.

In one embodiment of the invention, ion exchange chromatography may beused in binding mode or flow-through mode.

In certain embodiments, the Protein A chromatography step is conductedfirst, the anion exchange step is conducted second, and the cHA step isconducted third.

Removal of Additional Impurities

In addition to HMWA removal, cHA chromatography has been shown useful inremoving other impurities from antibody preparations. Other impuritiesthat may be removed by the cHA chromatography methods of the inventioninclude, but are not limited to, DNA, host cell protein, adventitiousviruses, and Protein A contaminants from prior purification steps.

In one embodiment of the invention, the invention is able to removeProtein A from the antibody preparation. In certain embodiments of thisinvention, the amount of Protein A present in the final preparation canbe reduced significantly, such as from 300 ppm to less than 1 ppm.

Administration and Method of Treatment

Formulations containing the SDAB molecules purified by the methodsdisclosed herein can be administered to a subject (e.g., a humansubject) alone or combination with a second agent, e.g., a secondtherapeutically or pharmacologically active agent, to treat or prevent(e.g., reduce or ameliorate one or more symptoms associated with) a TNFαassociated disorder, e.g., inflammatory or autoimmune disorders. Theterm “treating” refers to administering a therapy in an amount, manner,and/or mode effective to improve a condition, symptom, or parameterassociated with a disorder or to prevent progression of a disorder, toeither a statistically significant degree or to a degree detectable toone skilled in the art. An effective amount, manner, or mode can varydepending on the subject and may be tailored to the subject.

Non-limiting examples of immune disorders that can be treated include,but are not limited to, autoimmune disorders, e.g., arthritis (includingrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis, lupus-associated arthritis or ankylosingspondylitis), scleroderma, systemic lupus erythematosis, Sjogren'ssyndrome, vasculitis, multiple sclerosis, autoimmune thyroiditis,dermatitis (including atopic dermatitis and eczematous dermatitis),myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease,colitis, diabetes mellitus (type I); inflammatory conditions of, e.g.,the skin (e.g., psoriasis); acute inflammatory conditions (e.g.,endotoxemia, sepsis and septicaemia, toxic shock syndrome and infectiousdisease); transplant rejection and allergy. In one embodiment, the TNFαassociated disorder is, an arthritic disorder, e.g., a disorder chosenfrom one or more of rheumatoid arthritis, juvenile rheumatoid arthritis(RA) (e.g., moderate to severe rheumatoid arthritis), osteoarthritis,psoriatic arthritis, or ankylosing spondylitis, polyarticular juvenileidiopathic arthritis (JIA); or psoriasis, ulcerative colitis, Crohn'sdisease, inflammatory bowel disease, and/or multiple sclerosis.

In certain embodiments, the formulations include a second therapeuticagent. For example, for TNF-nanobodies, the second agent may be ananti-TNF antibody or TNF binding fragment thereof, wherein the secondTNF antibody has a different epitope specificity than the TNF-bindingSDAB molecule of the formulation. Other non-limiting examples of agentsthat can be co-formulated with TNF-binding SDAB include, for example, acytokine inhibitor, a growth factor inhibitor, an immunosuppressant, ananti-inflammatory agent, a metabolic inhibitor, an enzyme inhibitor, acytotoxic agent, and a cytostatic agent. In one embodiment, theadditional agent is a standard treatment for arthritis, including, butnot limited to, non-steroidal anti-inflammatory agents (NSAIDs);corticosteroids, including prednisolone, prednisone, cortisone, andtriamcinolone; and disease modifying anti-rheumatic drugs (DMARDs), suchas methotrexate, hydroxychloroquine (Plaquenil) and sulfasalazine,leflunomide (Arava®), tumor necrosis factor inhibitors, includingetanercept (Enbrel®), infliximab (Remicade®) (with or withoutmethotrexate), and adalimumab (Humira®), anti-CD20 antibody (e.g.,Rituxan®), soluble interleukin-1 receptor, such as anakinra (Kineret),gold, minocycline (Minocin®), penicillamine, and cytotoxic agents,including azathioprine, cyclophosphamide, and cyclosporine. Suchcombination therapies may advantageously utilize lower dosages of theadministered therapeutic agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies.

The formulations can be in the form of a liquid solution (e.g.,injectable and infusible solutions). Such compositions can beadministered by a parenteral mode (e.g., subcutaneous, intraperitoneal,or intramuscular injection), or by inhalation. The phrases “parenteraladministration” and “administered parenterally” as used herein meanmodes of administration other than enteral and topical administration,usually by injection, and include, subcutaneous or intramuscularadministration, as well as intravenous, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcuticular,subcapsular, subarachnoid, intraspinal, epidural and intrasternalinjection and infusion. In one embodiment, the formulations describedherein are administered subcutaneously.

Pharmaceutical formulations are sterile and stable under the conditionsof manufacture and storage. A pharmaceutical composition can also betested to insure it meets regulatory and industry standards foradministration.

A pharmaceutical formulation can be formulated as a solution,microemulsion, dispersion, liposome, or other ordered structure suitableto high protein concentration. Sterile injectable solutions can beprepared by incorporating an agent described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating anagent described herein into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

EXAMPLES

The following examples are offered for illustrative purposes only.

Example 1 Description of the ATN-103 Coding Sequence

ATN-103 is a trivalent nanobody molecule targeting TNFα and HSA.Nanobodies were isolated from a llama derived phage library by selectionon TNFα or HAS as described in WO 06/122786. Nanobodies were tested forspecific activity and TNF1 was chosen as the nanobody inhibitor of humanTNFα and ALB1 the human anti-HSA nanobody for half-life extension. TNF1and ALB1 were humanized by CDR grafting onto the closest human framework(DP51/DP53). During humanization of TNF1, 2 camelid residues wereretained (P84 and R103) and this version designated TNF30. Duringhumanization of ALB1, 7 camelid residues were retained (N16, N73, T76,P84, T93, 194 and 5103) and this version designated ALB8. Two TNF30nanobodies were each linked by a 9 amino-acid Glycine-Serine linker(Gly₄SerGly₃Ser (SEQ ID NO:9)) to a central ALB8 nanobody to give thetrivalent molecule designated herein as “ATN-103.” The amino acidsequence of ATN-103 is shown in FIG. 3 having the followingconfiguration TNF30-(Glycine-Serine linker)-ALB8-(Glycine-Serinelinker)-TNF30. In FIG. 2, complementarity determining regions (CDR) areunderlined (SEQ ID NOs:2-7). The predicted intramolecular disulfidebonds are illustrated by connections of the cysteine residues involved.The binding domains to TNF are shown in bold and the binding domain toHSA is shown in bold italics. The amino acid linkers connecting thesebinding domains are in italics. The signal peptide (⁻¹⁹MGW . . . VHS⁻¹)is also shown for the polypeptide chain.

Example 2 ATN-103 Purification Process

The ATN-103 purification process consists of two chromatographic stepsand three membrane filtration steps (see FIG. 3). All steps areperformed at room temperature unless indicated otherwise.

The principles, objectives, and descriptions of each purification stepare provided below.

MabSelect Protein A Affinity Chromatography and Low pH VirusInactivation

The primary objectives of the MabSelect™ Protein A chromatography stepinclude product capture from clarified cell-free conditioned medium andseparation of ATN-103 from process-derived impurities (e.g., host cellDNA and protein, medium components, and adventitious agents).

MabSelect Protein A is an affinity resin composed of a highlycross-linked agarose matrix that is covalently derivatized through athioether linkage with recombinant Protein A produced from Escherichiacoli (E. coli) fermentation.

The MabSelect Protein A column is equilibrated with Tris-buffered sodiumchloride solution and loaded with clarified cell-free conditioned medium(CM). All buffers run at 300 cm/hr. The equilibration buffer contains150 mM NaCl and 50 mM Tris at pH 7.5. ATN-103 binds to the MabSelect™Protein A resin and impurities flow through the column. The loaded resinis washed with a Tris-buffered sodium chloride solution (150 mM NaCl and50 mM Tris at pH 7.5) to further reduce the level of impurities,followed by a low concentration Tris buffer. The low concentration Triswashing buffer contains 10 mM NaCl and 10 mM Tris at pH 7.5. The boundproduct is eluted from the column with a low pH glycine buffer. The lowpH glycine elution buffer contains 10 mM NaCl and 50 mM glycine at pH3.0. The resin is regenerated and sanitized with a hydroxide solutionand then stored in a solution containing 16% ethanol. Multiple cycles ofthe MabSelect Protein A step can be generated from one harvest.

The product pool is held at pH≦3.8 for 1.5±0.5 h at 18° C. to 24° C. Thelow pH inactivation following the MabSelect™ Protein A column has beendesigned to inactivate enveloped viruses. The elution pool is thenneutralized with a concentrated HEPES buffer (2 mM HEPES at pH 9.0) andfiltered using depth and 0.2 μm filtration.

Macro-Prep Ceramic Hydroxyapatite Chromatography

The primary objectives of the Macro-Prep™ ceramic hydroxyapatite (cHA)step are the removal of high molecular weight aggregates (HMWA), leachedProtein A, and host cell-derived impurities, such as DNA and host cellproteins (HCPs).

Macro-Prep ceramic hydroxyapatite is an incompressible matrix composedof a hexagonally-crystalline lattice. Calcium, phosphate, and hydroxidemolecules comprise the matrix, with a stoichiometry of (Ca₅(PO₄)₃(OH))₂.The cHA resin is a multi-mode matrix capable of promoting cationexchange, anion exchange, and metal coordination interactions. Elutionof bound proteins is typically achieved with increases in salt orphosphate concentration.

The cHA column is first equilibrated with a buffer containing a highconcentration of sodium chloride followed by a buffer containing a lowconcentration of sodium chloride. The load to the cHA column is theneutralized MabSelect™ Protein A pool. After loading, the column iswashed with a low salt equilibration buffer and ATN-103 is recoveredusing a buffer containing higher salt concentration. After elution ofATN-103, HMW and other impurities are removed from the column at muchhigher salt and phosphate concentrations. The column is regenerated andthen stored in a sodium hydroxide solution.

Planova 20N Virus Retaining Filtration

The Planova 20N virus retaining filtration (VRF) step provides asignificant level of viral clearance for assurance of product safety byremoval of particles that may represent potential adventitious viralcontaminants.

The single-use Planova 20N VRF device is equilibrated cHA elution bufferand loaded with the Macro-Prep ceramic hydroxyapatite product pool. Theproduct is collected in the permeate stream. After the load isprocessed, a buffer flush is used to recover additional productremaining in the system.

Ultrafiltration/Diafiltration and Formulation

An ultrafiltration/diafiltration step (10 kDa MW cut off) is used toconcentrate and buffer exchange the VRF product into the formulationbuffer.

After equilibration of the membrane module, the load solution isinitially concentrated to a preset target volume and then diafilteredwith 14 mM Histidine pH 5.8 buffer. Following further concentration toapproximately 110 g/L, the pool is recovered from the system with ahistidine buffer flush to achieve a final protein target concentrationof approximately 90 g/L. A small volume (11.1% v/v) of concentratedstock solution (10 mM Histidine, 50% Sucrose and 0.1% Polysorbate 80) isadded to the product pool. The final drug substance (DS) obtained is 80g/L ATN-103 in 10 mM Histidine pH 6.0, 5% sucrose, 0.01% Polysorbate.

Final Filtration

The formulated drug substance is passed through a single-use 0.2 μmfilter to remove any potential adventitious microbial contaminants andparticulate material.

Example 3 Protein A Capture Comparison

The following Protein A based matrixes were evaluated for theircapacities to capture ATN-103: MabSelect™ (GE Healthcare), MabSelectXtra™ (GE Healthcare), ProSep® Va Ultra Plus (Millipore), and MabSelectSuRe™ (GE Healthcare). MabSelect™ uses Protein A ligand containingZ-domain and the resin backbone is more hydrophobic due to crosslinking. MabSelect Xtra™ uses the same ligand as MabSelect with 30%increased density and has smaller beads and larger pore size. ProSep® VaUltra Plus has glass based backbone and native Protein A ligand. It isdesigned for higher capacities at higher flow rates. MabSelect SuRe™binds Fc containing molecules (ATN-103 does not have an Fc region) andits novel ligand allows for greater caustic stability.

When a Protein A peak pool, which had been purified by MabSelect™, wasused as the loading material (pH=7.0, diluted to 1 g/L (expectedcondition media concentration)), ProSep® Va Ultra Plus showed thehighest binding capacity (16 g/L r) and the binding capacity ofMabSelect™ showed a 20% increase compared to previously demonstratedbinding capacity.

The impact of flow rate on Dynamic Binding Capacity (DBC) was examined.For MabSelect™, the binding capacity was 7.4 g/L r at 600 cm/hr,compared to 8.0 g/L r at 60 cm/hr. Similar trend was observed whenMabSelect Xtra™ and ProSep® Va Ultra Plus were tested. Thus, there wasminimal impact of flow rate on DBC under the tested conditions.

The effect of modifier on Protein A binding capacity was also examined.The results showed that the addition of 0.5 M Na₂SO₄ could increase DBCby enhancing hydrophobic interactions. For example, the binding capacityfor MabSelect™ (with CM) increased to 12.5 g/L r with the flow rate of150 cm/hr. Additional bound material was subsequently eluted in Na₂SO₄free solution. High amount of precipitation was detected in CM withNa₂SO₄. Similar results were observed when MabSelect Xtra™ (DBC=16 g/Lr) and ProSep® Va Ultra Plus (DBC=17.5 g/L r) were tested.

To examine the effect of PEG on Protein A binding capacity, 6% PEG (4000Da) was added into CM. Additional bound material was subsequently elutedin PEG free buffer. The results showed a slight increase in bindingcapacity and no precipitation was detected.

Example 4 ATN-103 MabSelect SuRe™ Evaluation

During the development of the MabSelect step MabSelect Sure™ was used inbinding experiments to gain a better understanding of the Protein Abinding mechanism to ATN-103. Unexpectedly, the ATN-103 bound toMabSelect Sure™ and required a solution with pH 4.5 to remove the boundproduct. MabSelect Sure is Protein A resin that was designed to bindonly molecules containing an Fc region such as antibodies. ATN-103 doesnot contain an Fc region. Later in development it was determined thatMabSelect Sure™ could bind up to 8 g/L resin of ATN-103 at 10%breakthrough of the initial concentration.

Example 5 ATN-103 Cation Exchange (CEX) Step Evaluation

A Cation Exchange (CEX) based capture step was evaluated in an effort toincrease the capacity of the capture column in the ATN-103 purificationprocess. By lowering the conductivity of the condition media (CM)between 12 and 9 mS/cm and titrating the pH to ≦4.3 a capacity of 40 g/Lr were observed. ATN-103 binds very weakly to the CEX medium due to thelow number of charge/mole at these pH levels. Because of this weakbinding, ATN-103 can be eluted from the CEX resin in low conductivitysolutions. The eluting conditions were generally ≦50 mM NaCl at pH 6.5to 7.0. The CEX column could also be eluted using the downstream cHAequilibration buffer.

CEX Capacity Screen

Four cation exchangers, Capto™ S (GE Heathcare), Fractogel® 503-(M) (EMDChemicals), Toyopearl® Gigacap S-650M (Tosoh Bioscience) and Poros® HS50 (Applied Biosystems), were tested for their binding capacities forATN-103. 0.75 mL CM TS2 with 10 uL resin (target 75 g/L resin) was usedin the screening. Columns were washed with buffer containing 50 mMsodium acetate at the same pH as the loading condition. Proteins wereeluted with buffer containing 1M NaCl (pH 5.5). The bound mass wasmeasured by spectrophotometry at A280. Columns were subsequentlystripped with buffer containing 1M NaCl and Urea. Spectrophotometrymeasured at A280 showed that no significant mass was bound afterstripping. Up to about 25 g/L r of binding capacity was observed for allresins studied. Capto™ S and Toyopearl® Gigacap S-650M showed relativelyweaker binding, and Fractogel® 503-(M) and Poros® HS 50 showed tighterbinding. This study indicates that elution by pH with very lowconductivity conditions is possible.

CEX Binding Capacity

CM were titrated to pH4.0 and diluted 3:1 with Rodi water (3 parts CMwas added to 1 part Rodi) to 0.75 total dilution (8 mS/cm load). Bindingcapacities of Capto™ S, Toyopearl® Gigacap S-650M, Poros® HS 50 andFractogel® 503-(M) were initially measured for a target binding capacityof greater than 40 g/L using 2 mL column (0.5 cm×10 cm). The bindingcapacities of Capto™ S, Toyopearl® Gigacap S-650M and Fractogel® 503-(M)were further determined at various conditions within a pH range from 4.0to 4.3. For example, the binding capacity for Capto™ S was measured atpH 4.3, 9 mS/cm; pH 4.0, 11 mS/cm, pH 4.0, 9 mS/cm, and pH 4.1, 8 mS/cm,respectively. The binding capacity for Toyopearl® Gigacap S-650M wasmeasured at pH 4.3, 9 mS/cm and pH 4.1, 8 mS/cm. The binding capacityfor Fractogel® 503-(M) was measured at pH 4.3, 12 mS/cm; pH 4.3, 9mS/cm; pH 4.0, 12 mS/cm, pH 4.1, 9 mS/cm, and pH 4.0, 9 mS/cm,respectively. The results showed that Fractogel® 503-(M) could be usedwithout diluting CM with water. Without dilution Fractogel® 503-(M)showed good binding capacity between pH 4.0 and 4.3 (DBC range: 25to >40 g/L r). With 23% dilution, Fractogel® 503-(M) showed excellentcapacity between pH 4.0 and 4.3 (DBC range: 40 to >50 g/L r).

CEX Elution Approach

Gradient elutions were performed as described in the experiments for CEXbinding capacity. Citrate buffer was used for pH elution. Capto™ S,Toyopearl® Gigacap S-650M, Fractogel® SO3-(M) and Poros® HS 50 weretested. Size exclusion chromatography (SEC) elution showed high purityof ATN-103 and low level of HMW and LMW species. The degree of purity isat least comparable to that of the Protein A eluted materials. HTSscreen can be designed with the information obtained from the gradientelutions.

Example 6 High Concentration Ultrafiltration/Diafiltration andFormulation Example 6.1 High Concentration Ultrafiltration/Diafiltrationand Formulation

An ultrafiltration/diafiltration step (10 kDa MW cut off) is used toconcentrate and buffer exchange the VRF product into the formulationbuffer.

After equilibration of the membrane module, the load solution isinitially concentrated to a preset target volume and then diafilteredwith 28 mM Histidine pH 5.8 buffer. Following further concentration toapproximately 200 g/L, the pool is recovered from the system with ahistidine buffer flush to achieve a final protein target concentrationof approximately 150 g/L. A small volume (17.6% v/v) of concentratedstock solution (20 mM Histidine, 50% Sucrose and 0.0667% Polysorbate 80)is added to the product pool 80. The final DS obtained is 80 g/L ATN-103in 10 mM Histidine pH 6.0, 5% sucrose, 0.01% PS80. The final DS obtainedis 125 g/L ATN-103 in 20 mM Histidine pH 6.0, 7.5% sucrose, 0.01%Polysorbate.

Example 6.2 High Concentration Ultrafiltration/Diafiltration

An ultrafiltration/diafiltration step (10 kDa MW cut off) was used toconcentrate and buffer exchange the VRF product into the formulationbuffer.

After equilibration of the membrane module, the load solution wasinitially concentrated to approximately 40 g/L and then diafiltered with30 mM Histidine, pH 5.8, 8.5% Sucrose buffer. Following furtherconcentration to approximately 300 g/L, the pool was recovered from thesystem with diafiltration buffer to achieve a final protein targetconcentration of approximately 175 g/L.

Example 6.3 High Concentration Ultrafiltration/Diafiltration andFormulation

An ultrafiltration/diafiltration step (10 kDa MW cut off) was used toconcentrate and buffer exchange the VRF product into the formulationbuffer.

After equilibration of the membrane module, the load solution wasinitially concentrated to approximately 40 g/L and then diafiltered with30 mM Histidine pH 5.8 buffer. Following further concentration toapproximately 320 g/L, the pool was recovered from the system withdiafiltration buffer to achieve a final protein target concentration ofapproximately 210 g/L.

If the DS target was >=200 g/L, a combination of Example 6.2 and Example6.3 would be performed as follows: An ultrafiltration/diafiltration step(10 kDa MW cut off) could be used to concentrate and buffer exchange theVRF product into the formulation buffer. After equilibration of themembrane module, the load solution could be initially concentrated to 40g/L and then diafiltered with a histidine sucrose buffer. Followingfurther concentration to approximately 320 g/L, the pool would berecovered from the system with diafiltration buffer to achieve a finalprotein target concentration of approximately >=200 g/L where thesucrose is already present. Then the formulation spike would be 1.01%v/v so the UF pool won't get significantly diluted and the final DSwould be >=200 g/L.

EQUIVALENTS

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

1. A method of separating or purifying a single domain antigen binding(SDAB) molecule that comprises one or more nanobody molecules from amixture containing the SDAB molecule and one or more contaminants,comprising: contacting the mixture with a Protein A-based or a cationexchange-based support, under conditions that allow the SDAB molecule tobind or absorb to the support; removing one or more contaminants; andselectively eluting the SDAB molecule from the support, wherein saidSDAB molecule does not have a complementary antibody variable domain oran immunoglobulin Fc region.
 2. The method of claim 1, wherein the stepof removing one or more of the contaminants comprises washing the boundsupport under conditions where the SDAB molecule remains bound to thesupport.
 3. The method of claim 1, wherein the mixture is contacted witha Protein A-based support.
 4. The method of claim 3, wherein the step ofremoving one or more of the contaminants comprises washing the boundsupport with at least one Protein A washing buffer, wherein said ProteinA washing buffer comprises about 100 to about 175 mM NaCl and about 40to about 60 mM Tris at pH ranging from about 7 to 7.5.
 5. The method ofclaim 4, wherein the step of selectively eluting the SDAB molecule fromthe support comprises eluting the adsorbed SDAB molecule with at leastone Protein A elution buffer, wherein said Protein A eluting buffercomprises about 5 to about 50 mM NaCl and about 5 mM to about 100 mMglycine at pH 4.0 or less.
 6. The method of claim 1, further comprisingone or more of: hydroxyapatite chromatography, cation exchangechromatography, affinity chromatography, size exclusion chromatography,hydrophobic interaction chromatography, metal affinity chromatography,diafiltration, ultrafiltration, viral inactivation or viral removalfiltration.
 7. The method of claim 6, further comprising contacting themixture with a hydroxyapatite resin and selectively eluting the SDABmolecule from the hydroxyapatite resin.
 8. The method of claim 6,further includes contacting the mixture with a cation exchange column,and selectively eluting the SDAB molecule from the column.
 9. The methodof claim 1, wherein the SDAB molecule is a recombinant protein producedin a mammalian host cell.
 10. The method of claim 9, wherein themammalian host cell is a CHO cell.
 11. The method of claim 1, whereinthe contaminants in the mixture comprise one or more of high molecularweight protein aggregates, host cell proteins, DNA, or leached ProteinA.
 12. The method of claim 1, wherein the SDAB molecule is purified toat least 90% higher purity.
 13. The method of claim 3, wherein theProtein A-based support comprises a resin of immobilized recombinant orisolated full length Staphylococcal Protein A (SpA), or a functionalvariant thereof.
 14. The method of claim 13, wherein the full length SpAcomprises the amino acid sequence of SEQ ID NO:11 shown in FIG. 4A, oran amino acid sequence at least 90% identical thereto.
 15. The method ofclaim 13, wherein the functional variant of SpA comprises at leastdomain B of SpA, or a variant of domain B, having one or moresubstituted asparagine residues.
 16. The method of claim 15, wherein thefunctional variant of SpA comprises the amino acid sequence of SEQ IDNO:12 shown in FIG. 4B, or an amino acid sequence at least 90% identicalthereto.
 17. The method of claim 1, wherein the mixture is contactedwith a cation exchange-based support.
 18. The method of claim 17,wherein the method comprises: contacting the mixture containing the SDABmolecule and one or more contaminants with a cation exchange (CEX)resin, wherein the CEX resin shows a capacity of at least about 40 g/L;allowing the SDAB molecule to flow through the support, washing thesupport with at least one cation exchange washing buffer; and elutingthe SDAB molecule with an elution buffer.
 19. The method of claim 18,wherein the conductivity of the condition media (CM) used to load thecolumn is between about 12 and 9 mS/cm and the pH of the loadingconditions is adjusted to be equal to or less than about 4.5.
 20. Themethod of claim 19, wherein the elution buffer is equal to about 50 mMsodium chloride or less, and has a pH of about 5.5 to 7.2.
 21. Themethod of claim 18, further comprising further comprising contacting themixture with a hydroxyapatite resin and selectively eluting the SDABmolecule from the hydroxyapatite resin.
 22. The method of claim 21,pre-treating the mixture with an equilibration buffer and allowing thepre-treated mixture to flow through a hydroxyapatite resin.
 23. Themethod of claim 22, wherein the contacting step of the mixture with ahydroxyapatite resin and the elution steps are effected in buffersolutions comprising about 1 to 20 mM sodium phosphate and from about0.2 to 2.5 M sodium chloride, in a pH from about 6.4 to 7.6.
 24. Themethod of claim 22, wherein the equilibration buffer comprise about 1 to20 mM sodium phosphate, from about 0.01 to 2.0 M sodium chloride, fromabout 0 to 200 mM arginine, from about 0 to 200 mM HEPES, in a pH fromabout 6.2 to 8.0.
 25. The method of claim 1, wherein the purified SDABmolecule contains less than 10% high molecular weight aggregates.
 26. Amethod of separating or purifying a single domain antigen binding (SDAB)molecule that comprises one or more nanobody molecules from a mixturecontaining the SDAB molecule and one or more contaminants, comprising:contacting the mixture with a Protein A-based support, under conditionsthat allow the SDAB molecule to bind or absorb to the Protein A-basedsupport; removing one or more contaminants; selectively eluting the SDABmolecule from the support, thereby obtaining an eluted SDAB moleculepreparation; contacting the eluted SDAB molecule preparation with ahydroxyapatite resin; and selectively eluting the SDAB molecule from thehydroxyapatite resin, wherein said SDAB molecule does not have acomplementary antibody variable domain or an immunoglobulin Fc region.27. A method of separating or purifying a single domain antigen binding(SDAB) molecule that comprises one or more nanobody molecules from amixture containing the SDAB molecule and one or more contaminants,comprising: contacting the mixture with a Protein A-based support, underconditions that allow the SDAB molecule to bind or absorb to the ProteinA-based support; removing one or more contaminants; selectively elutingthe SDAB molecule from the support, thereby obtaining an eluted SDABmolecule preparation; pre-treating the eluted SDAB molecule preparationwith an equilibration buffer; and allowing the pre-treated mixture toflow through a hydroxyapatite resin, wherein said SDAB molecule does nothave a complementary antibody variable domain or an immunoglobulin Fcregion.
 28. A method of separating or purifying a single domain antigenbinding (SDAB) molecule that comprises one or more nanobody moleculesfrom a mixture containing the SDAB molecule and one or morecontaminants, comprising: contacting the mixture containing the SDABmolecule and one or more contaminants with a cation exchange support,allowing the SDAB molecule to flow through the support, washing thesupport with at least one cation exchange washing buffer; removing oneor more contaminants; selectively eluting the SDAB molecule from thesupport, thereby obtaining an eluted SDAB molecule preparation;contacting the eluted SDAB molecule preparation with a hydroxyapatiteresin; and selectively eluting the SDAB molecule from the hydroxyapatiteresin, wherein said SDAB molecule does not have a complementary antibodyvariable domain or an immunoglobulin Fc region.
 29. The method of claim1, wherein the SDAB molecule is a single chain polypeptide, comprisingat least one immunoglobulin variable domain.
 30. The method of claim 29,wherein the SDAB molecule comprises at least one immunoglobulin variabledomain from an antibody naturally devoid of light chains.
 31. The methodof claim 30, wherein the antibody is a camelid antibody.
 32. The methodof claim 29, wherein the SDAB molecule is a monovalent, bivalent, ortrivalent molecule.
 33. The method of claim 29, wherein the SDABmolecule is a monospecific, bispecific, or trispecific molecule.
 34. Themethod of claim 29, wherein the SDAB molecule comprises one or morenanobody molecules that are recombinant, CDR-grafted, humanized,camelized, de-immunized, and/or in vitro selected by phage display. 35.The method of claim 29, wherein the SDAB molecule binds to one or moretarget antigens selected from the group consisting of a cytokine, growthfactor and a serum protein.
 36. The method of claim 35, wherein the SDABmolecule binds tumor necrosis factor α (TNF α).
 37. The method of claim35, wherein the SDAB molecule binds to human serum albumin (HSA). 38.The method of claim 29, wherein the SDAB molecule is a trivalent,bispecific molecule composed of a single chain polypeptide fusion of twocamelid variable regions that bind to TNF α, and one camelid variableregion that binds to HSA.
 39. The method of claim 38, wherein the SDABmolecule is arranged in the following order from N- to C-terminus:Anti-TNFα SDAB molecule—anti-HSA SDAB molecule—anti-TNFα SDAB molecule.40. The method of claim 1, wherein the SDAB molecule comprises the aminoacid sequence of SEQ ID NO:1, or an amino acid sequence at least 90%identical to thereto.
 41. The method of claim 1, wherein the SDABmolecule comprises at least one of the SDAB molecule that binds to TNFαand comprises three CDRs having the amino sequence: SEQ ID NO:2 (DYWMY(CDR1)), SEQ ID NO:3 (EINTNGLITKYPDSVKG (CDR2)) and SEQ ID NO:4 (SPSGFN(CDR3)), or having a CDR that differs by fewer than 2 conservative aminoacid substitutions from one of said CDRs.
 42. The method of claim 1,wherein the SDAB molecule comprises at least one of the SDAB moleculethat binds to TNFα and comprises a variable region having the amino acidsequence from about amino acids 1 to 115 of SEQ ID NO:1 shown in FIG. 2,or an amino acid sequence at least 90% identical to the amino acidsequence of SEQ ID NO:1 shown in FIG.
 2. 43. The method of claim 41,wherein the SDAB molecule comprises at least one of the SDAB moleculethat binds to HSA and comprises three CDRs having the amino sequence:SFGMS (CDR1; SEQ ID NO:5), SISGSGSDTLYADSVKG (CDR2; SEQ ID NO:6) and/orGGSLSR (CDR3; SEQ ID NO:7), or having a CDR that differs by fewer than 2conservative amino acid substitutions from one of said CDRs.
 44. Themethod of claim 43, wherein the SDAB molecule comprises at least one ofthe SDAB molecule that binds to HSA and comprises a variable regionhaving the amino acid sequence from about amino acids 125 to 239 of SEQID NO:1 shown in FIG. 2, or an amino acid sequence at least 90%identical to the amino acid sequence of SEQ ID NO:1 shown in FIG.
 2. 46.The method of claim 39, wherein two or more of the SDAB molecules arefused with a linking group that comprises at least five, seven eight,nine, ten, twelve, or fifteen glycine and serine residues.
 47. Themethod of claim 46, wherein two or more of the SDAB molecules are fusedwith a linking group comprising the amino acid sequence of SEQ ID NO:9((Gly)₄-Ser-(Gly)₃-Ser).
 48. A method or process of providing arecombinant single domain antigen binding (SDAB) molecule that includesone or more nanobody molecules, comprising: providing a mammalian hostcell comprising a nucleic acid that encodes the recombinant SDABmolecule; maintaining the host cell under conditions in which therecombinant SDAB molecule, is expressed; obtaining a mixture of therecombinant SDAB molecule and one or more contaminants; purifying orseparating the recombinant SDAB molecule from said mixture using ProteinA-based or a cation exchange based chromatography, wherein saidpurifying or separating step comprises contacting the mixture with thesupport, under conditions that allow the SDAB molecule to bind or absorbto the support; removing one or more contaminants; and selectivelyeluting the SDAB molecule from the support, thereby obtaining an elutedSDAB molecule preparation, wherein said SDAB molecule does not have acomplementary antibody variable domain or an immunoglobulin Fc region.49. The method of claim 48, further comprising subjecting the elutedSDAB molecule preparation to one or more of hydroxyapatitechromatography, affinity chromatography, size exclusion chromatography,hydrophobic interaction chromatography, metal affinity chromatography,diafiltration, ultrafiltration, or viral removal filtration.
 50. Themethod of claim 48, further comprising preparing a formulation of therecombinant SDAB molecule as a pharmaceutical composition.
 51. Themethod of claim 1 or 18, further comprising concentrated the eluted SDABmolecule to a preselected target volume.
 52. The method of claim 51,wherein the concentration step is carried out by performing anultrafiltration/diafiltration step in the presence of a Histidine bufferor a Tris buffer.
 53. The method of claim 51, wherein the SDAB moleculeis concentrated to at least about 80 g/L to 350 g/L.
 54. An SDABmolecule purified by the method of claim
 1. 55. A pharmaceuticalcomposition comprising the SDAB molecule of claim
 54. 56. A method oftreating or preventing a disease in a subject comprising administeringto the subject the SDAB molecule of claim 54 in an amount effective totreat or prevent the disease.